Methods for determining ring number in carotenoids by lycopene epsilon-cyclasses and uses thereof

ABSTRACT

The invention relates to methods for mapping and characterizing catalytic domains in enzymes, preferably plant enzymes and those enzymes within the carotene synthesis family and more specifically ε-cyclase enzymes regulating formation of ε,ε-carotene. The methods include reverse PCR and site-directed mutagenesis for generating chimera and truncations or site-directed mutations of enzymes, respectively. These chimera, truncations or site directed mutants of ε-cyclase enzymes are useful in the characterization of the sequence residues conferring catalytic domains for the enzymes, and more specifically, the identification of single residues regulating catalytic activity for enzymes that are important in plant growth and photosynthesis. Chimeric enzymes generated by the methods of the invention can also be used to create transgenci hosts which are augmentated in their expression of specific carotene products.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §1.119(e) to provisional application serial No. 60/261,473, filed Jan. 12, 2001. The contents of this application are hereby incorporated by reference.

FEDERALLY-SPONSORED RESEARCH

[0002] The research described herein was supported by a grant from the National Science Foundation (MCB9631257). The government has certain rights to this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention provides methods for mapping catalytic domains of eukaryotic or prokaryotic enzymes of carotenoid biosynthesis and metabolism. The present invention also provides polynucleotides encoding chimeric enzymes, truncated enzymes and site-directed mutants of enzymes of carotenoid biosynthesis and metabolism and their use in identifying catalytic domains of the enzymes. The invention provides methods for obtaining transgenic hosts augmented in their expression of bicyclic-ε-carotene by transformation with polynucleotide constructs encoding chimeric enzymes. The present invention also describes the amino acid sequence of the catalytic domain for an ε-cyclase which catalyzes lycopene into bicyclic-ε-carotene.

[0005] 2. Description of the Related Art

[0006] Carotenoids with cyclic end groups are present in the photosynthetic reaction complexes of plants, algae, and cyanobacteria (1, 2). These lipid-soluble isoprenoid pigments protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments (3, 4, 5). The cyclization of the linear, pink carotenoid lycopene (FIG. 1), is a pivotal branch point in the pathway of carotenoid biosynthesis in green plants. Two types of cyclic end groups and derivatives thereof, are commonly found in carotenoids of plants: β and ε rings. These two end groups differ only in the position of the double bond within the cyclohexene ring (FIG. 1). Carotenoids with two β rings are ubiquitous (1, 2) and include β-carotene and zeaxanthin, pigments thought to serve primarily in protecting against photo-oxidation and/or in dissipation of excess light energy. Carotenoids with one β and one ε ring are also common in plants and include lutein, the predominant carotenoid in the light-harvesting antenna of most green plants. Carotenoids with two ε rings (ε, ε-carotene) are not commonly found, other than in trace amounts, in plants and algae (1).

[0007] The symmetrical bicyclic, yellow carotenoid pigment, ε,ε-carotene, is associated with the photosynthetic apparatus in oxygenic photosynthetic organisms and plays a vital role in protecting against potentially lethal photo-oxidative damage. Accordingly, these compounds may have widespread industrial applications in promoting plant growth and photosynthesis for large-scale agricultural operations. [to modify carotenoid colored plants tissues] Epsilon, epsilon-carotene may also have commercial use as food dyes and colorings as well as a pharmaceutical use as a chemopreventative agent (31).

[0008] Romaine lettuce is one of the rare plant species that produces an abundance of an ε,ε-carotenoid, the dihydroxy ε,ε-carotenoid lactucaxanthin. The present Inventors previously described the isolation and characterization of a gene encoding the lycopene epsilon cyclase from lettuce which forms ε,ε-carotene from lycopene, and found that the enzyme shares about 65% sequence identity with an Arabidopsis cyclase gene, lycopene epsilon cyclase (PCT/US99/10461, which is incorporated by reference herein in its entirety). The lettuce enzyme adds two epsilon rings to lycopene to form ε,ε-carotene, whereas the Arabidopsis enzyme adds only one epsilon ring to form the monocyclic δ-carotene, ε,ψ-carotene.

[0009] The previously described methods for producing carotenoids with two epsilon rings are deficient in identifying the molecular basis for ε-cyclase catalytic activity effecting bicyclic ε-ring additions to lycopene versus the addition of only a single epsilon ring. Accordingly, there exists a need in the art for understanding the sequence identity of catalytic domains for this family of plant enzymes which are essential to plant growth and photosynthesis, and methods for identifying the same.

[0010] With such sequence information at hand, novel enzymes, which participate in the formation of ε,ε-carotene, can be created by replacing portions of one gene from one species with an analogous sequence of a related gene from another species. Through transfection of host cells with any such chimeric gene constructs or constructs containing site-directed mutants of genes, expression of recombinant carotenoid-synthesizing enzymes can be used to augment production of ε,ε-carotene in cells, which otherwise produce little or no ε,ε-carotene.

SUMMARY OF THE INVENTION

[0011] Accordingly, the present invention provides a method for mapping catalytic domains of eukaryotic enzymes of carotenoid biosynthesis and metabolism by chimeric enzymes.

[0012] The present invention provides a method for mapping catalytic domains of eukaryotic enzymes of carotenoid biosynthesis and metabolism by truncation of the enzymes.

[0013] The present invention provides a method for mapping catalytic domains of eukaryotic enzymes of carotenoid biosynthesis and metabolism by site-directed mutation of the enzymes.

[0014] The present invention also relates to polynucleotides encoding chimeric enzymes, truncated enzymes and site-directed mutants of enzymes of carotenoid biosynthesis and metabolism.

[0015] The present invention provides a method for augmenting the production of ε,ε-carotene in transformed host cells by transfection with constructs encoding chimeric carotenoid synthesizing enzymes from different sources.

[0016] The present invention relates to an amino acid sequence for a catalytic domain of ε-cyclase for catalyzing lycopene into bicyclic-ε-carotene.

[0017] Finally, the present invention describes the amino acid sequence for a catalytic domain of a lettuce ε-cyclase, and permissible amino acid substitutions.

[0018] These and other objects of the present invention have been realized by the present inventors as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

[0020]FIG. 1 depicts possible routes of synthesis of cyclic carotenoids and some common plant and algal xanthophylls (oxycarotenoids) from lycopene.

[0021]FIG. 2 describes a PCR strategy for constructing a chimera of lettuce and Arabidopsis lycopene ε-cyclase cDNAs.

[0022]FIG. 3 depicts HPLC elution profiles and absorption spectra of carotenoids produced in a lycopene-accumulating E. coli strain (14) in the presence of cDNAs encoding a lettuce lycopene ε-cyclase (panel A) and an Arabidopsis lycopene ε-cyclase (panel B).

[0023]FIG. 4 shows the alignment of deduced amino acid sequences of Arabidopsis (At) and lettuce (Ls) lycopene ε-cyclases. Residues identical for both sequences in a given position are in white text on a black background. A region of interest is underlined.

[0024]FIG. 5 is a schematic illustration of truncated and chimeric lycopene ε-cyclase cDNAs.

[0025]FIG. 6 depicts the catalytic domain for determining the number of ε rings added to lycopene by Arabidopsis and lettuce lycopene ε-cyclase. The catalytic domain was mapped to a six amino acid region defined by the residues ALIVQF in the Arabidopsis ε-cyclase and SHIVLM in the lettuce ε-cyclase (see FIGS. 4 and 5). Deduced amino acid sequences of lycopene mono-ε-cyclases from tomato (9), marigold (18) and potato are also displayed for this region. Similarly conserved residues are shown in black text on a gray background. Three amino acid residues in the lettuce bi-ε-cyclase that differ significantly from those in the known mono-ε-cyclases are in white text on a black background. Sequences of an Arabidopsis LCYb (a bicyclase introducing two beta rings) and an Adonis LCYe of mixed function are displayed below the lettuce LCYe with two residues of interest shown in white text on a black background.

[0026]FIG. 7 shows a neighbor-joining tree for deduced amino acid sequences of plant lycopene β- and ε-cyclases (LCYb and LCYe; 7, 9, 18) and of the related plant enzymes capsanthin-capsorubin synthase (CCS; 21) and neoxanthin synthase (NSY; 22, 23). Reactions catalyzed by the various enzymes are illustrated below the tree.

[0027]FIG. 8 depicts an alignment of deduced amino acid sequences of plant β- and ε-cyclases, neoxanthin synthases (NSY) and capsanthin capsorubin synthase (CCS) enzymes, and a cyanobacterial (Synechococcus PCC7942) β-cyclase.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Recently, the present inventors characterized a romaine lettuce gene encoding the enzyme, lycopene ε-cyclase, which catalyzes the addition of two epsilon rings to lycopene forming ε,ε-carotene. For the green plant Arabidopsis thaliana, two distantly related single copy genes encode enzymes that catalyze the introduction of the β and ε rings to lycopene (7). The Arabidopsis enzymes share 36% homology at the amino acid level. The Arabidopsis lycopene β-cyclase (LCYb) adds two rings to the symmetrical lycopene to form the bicyclic β-carotene (β,β-carotene; FIGS. 1 and 7). On the other hand, the lycopene ε-cyclase (LCYe) of Arabidopsis adds one ε ring to form the monocyclic δ-carotene (ε,ψ-carotene; FIGS. 1 and 7). These differences in function provide a mechanism for adjusting the proportions of β,β-carotenoids that are essential for photoprotection or the β,ε-carotenoids that serve primarily to capture light energy for photosynthesis, while at the same time preventing formation of carotenoids with two ε rings in Arabidopsis (7, 8, 9).

[0029] These different catalytic properties for enzymes within the related family of carotenoid ε-cyclase genes from different plant sources, lead the inventors to develop different strategies for mapping the catalytic domain of the ε-cyclase enzyme from lettuce and Arabidopsis as well as potato, tomato, Adonis and marigold (see FIG. 6). The catalytic domains were then sequenced in order to identify the amino acid residues, which confer the ring number determination for the respective enzymes.

[0030] Accordingly, the present invention provides different methods for mapping catalytic domains of eukaryotic or prokaryotic enzymes, more preferably, enzymes related to carotenoid biosynthesis and metabolism in plants.

[0031] A method of the present invention for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic a carotenoid-synthesizing enzyme, comprises

[0032] a) providing a first polynucleotide encoding a full-length enzyme and a second polynucleotide encoding a full-length enzyme, each polynucleotide being sub-cloned in tandem into a vector;

[0033] b) providing a first primer for hybridizing to the first polynucleotide and a second primer for hybridizing to the second polynucleotide;

[0034] c) performing an inverse polymerase chain reaction using the first and the second primer and the vector to obtain a construct containing a chimeric polynucleotide containing a 5′ end of the first polynucleotide and a 3′ end of the second polynucleotide;

[0035] d) repeating steps b) and c) with a plurality of different first primers and a plurality of different second primers for obtaining a plurality of constructs containing different chimeric polynucleotides for scanning along the encoded amino acid sequence one amino acid at a time;

[0036] e) transfecting a host cell with one or more of the plurality of constructs and growing the host cell under conditions for expressing chimeric proteins encoded by the chimeric polynucleotides;

[0037] f) performing enzyme catalysis with the chimeric proteins on an enzyme-specific substrate in the host cell,

[0038] wherein the substrate is preferably, a symmetrical carotenoid such as lycopene; and

[0039] g) identifying the enzyme-catalyzing domain encoded by the chimeric proteins by identification of at least one carotenoid compound from the enzyme catalysis of step f).

[0040] The length of the fragment for the first and the second polynucleotide, respectively, can vary with any such chimeric construct, and is limited by the primer pairs used to generate the construct. Thus, the extent to which either the first or second polynucleotide is included in the construct can be used to determine both the position and sequence identity for the catalytic domain of the given gene. Examples of chimeric gene constructs include a vector containing the first half (5′) of a romaine lettuce cyclase gene in combination with the second half (3′) of another plant cyclase gene, such as the Arabidopsis or potato gene, or the first half of an Arabidopsis or other mono-epsilon cyclase gene in combination with the second half (3′) of a lettuce cyclase gene. In some examples, chimeric constructs were obtained where the catalytic domain of one polynucleotide was replaced with that of the other polynucleotide.

[0041]FIG. 5 depicts several chimeric polynucleotides generated by the above-described method wherein a catalytic domain for one enzyme was replaced with the domain from a related gene of a different source. Accordingly, these chimeric constructs were used both to map the catalytic domains for the enzymes of the invention and to identify the residues responsible for regulating specific enzymatic activity. More specifically, the chimeric constructs were designed to identify residues, which confer ring number determinants.

[0042] The present inventors have identified the molecular basis for ε-cyclase catalytic activity effecting bicyclic ε-ring additions to lycopene. To this end, various ε cyclase chimera were constructed from an ε-cyclase cDNA from romaine lettuce, a plant known to accumulate substantial amounts of a carotenoid with two ε rings (10, 11), and an ε-cyclase cDNA from Arabidopsis, which adds only a single E-ring to lycopene. These chimeric ε-cyclases were assayed for their ability to convert lycopene into the bicyclic ε-carotene in a strain of Escherichia coli engineered to accumulate lycopene. Through this approach, a catalytic region for each of the Arabidopsis and lettuce ε-cyclases was defined as being integral to the determination of ring number. Additionally, by using this approach, chimera were obtained wherein the catalytic domain of Arabidopsis was switched for the catalytic domain of lettuce and vice versa.

[0043] Sources of enzyme include those eukaryotic and prokaryotic organisms, which produce carotenoids including plants, algae, yeasts, fungi, cyanobacteria and other photosynthetic bacteria. Preferred plants are lettuce, Arabidopsis, potato, Adonis, marigold or tomato. Preferred algae are of the genus dunaliella and haematococcos.

[0044] Enzymes include but are not limited to low abundance, membrane-associated enzymes, members of the carotenoid cyclase family as well as enzymes that catalyze reactions that utilize symmetrical substrates in the carotenoid pathway such as phytoene saturase, beta carotene hydroxylase, epsilon, epsilon-carotene, zeaxanthin and violaxanthin.

[0045] The low abundance, membrane-associated enzymes include phytoene desaturase, beta ring hydroxylase, epsilon ring hydroxylase, violaxanthin de-epoxidase and beta carotene ketolase.

[0046] The carotenoid cyclase enzymes include β- and ε-cyclases, neoxanthin synthases, capxanthin capsorubin synthases, and a cyanobacterial (Synechococcus PCC7942) β-cyclase. Epsilon-cyclase is a most preferred embodiment.

[0047] Suitable vectors contain a eukaryotic or prokaryotic gene encoding an enzymatic domain catalyzing a reaction of carotenoid biosynthesis or metabolism. Alternatively, the vectors contain a chimeric polynucleotide encoding a chimeric enzyme containing an enzymatic domain from a related gene from another source. Any such vector contains a suitable promotor for the host, and can be constructed using techniques well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Suitable vectors for prokaryotic expression include pACYC184, pUC119, pBR322 (New England Biolabs, Beverley, Mass.), pTrcHis (Invitrogen), Bluescript SK (Stratagene), pET28 (Novagen) and derivatives thereof.

[0048] The present vectors can additionally contain regulatory elements such as promoters, repressors, selectable markers such as antibiotic resistance genes, etc.

[0049] The first and the second primer are designed to hybridize anywhere within the full-length sequence for the respective polynucleotide including non-coding regions. Primers for obtaining chimeric polynucleotide constructs by an inverse polymerase chain reaction as described herein below and depicted in FIG. 2, were designed so that the first primer recognizing the first polynucleotide hybridizes in the 3′-5′ direction and the second primer recognizing the second polynucleotide hybridizes in the 5′-3′ direction. Preferred first primers include but are not limited to SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17 and 19. Preferred second primers include but are not limited to SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18 and 20.

[0050] As shown in Table 1, preferred pairing for the first and second primers includes but is not limited to SEQ ID NOS 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; SEQ ID NOS: 15 and 16; SEQ ID NOS: 17 and 18; and SEQ ID NOS: 19 and 20.

[0051] Host systems according to the present invention can comprise any organism that already produces carotenoids or which has been genetically engineered to produce carotenoids. Suitable hosts include E. coli, cyanobacteria such as Synechococcus and Synechocystis, algae and plant cells. E. coli is a preferred embodiment.

[0052] Transformation of the hosts with the chimeric constructs or vectors of the present invention can be done using standard techniques well known in the art, and incorporated herein by reference.

[0053] Transformed E. coli can be cultured using conventional techniques. The culture broth preferably contains antibiotics to select and maintain plasmids though the construct may alternatively be introduced within the genome where no antibiotic maintenance is required. Suitable antibiotics include penicillin, ampicillin, chloramphenicol, etc. Culturing is typically conducted at 15-45° C., preferably at room temperature (16-28° C.) for 12 hours to 7 days. E coli cultures are plated and the plates are screened visually for colonies with a different color than the colonies of the host E. coli transformed with an empty vector.

[0054] The mapping method of the instant invention encompasses identifying catalytic domains of enzymes based in the enzyme catalyzing a reaction on a suitable substrate and resulting in the formation of a different product or a modified substrate. A preferred enzyme and substrate combination is ε-cyclase and lycopene, respectively, and the result of the enzyme catalysis reaction is the formation of a carotene compound such as the introduction of epsilon and beta rings by e-cyclase (7).

[0055] Methods for detecting formation of carotenoid compounds include but are not limited to calorimetric assays, HPLC, TLC, mass spectrometry and absorption spectroscopy. Colorimetric assays, more preferably color complementation assays, were used to identify the formation of a different carotenes generated from the enzyme catalysis reactions of the chimeric enzymes of the invention on a lycopene substrate. For confirmation, HPLC and absorption spectroscopy were used to identify the carotenoids produced in lycopene-accumulating E. coli transfected with different chimeric constructs. The above-mentioned assay methods are not limited to detection of carotenoids in E. coli, but include any host cell expressing lycopene and capable of producing carotenoids, or otherwise transfected with any one of the chimeric constructs of the invention.

[0056] Carotenoids include but are not limited to ε,ε-carotene, ε,ψ-carotene, or a combination thereof, and β,β-carotene.

[0057] In a preferred embodiment, the addition of epsilon cyclic end groups to the pink-colored lycopene results in the formation of products that are yellow or yellow-orange in color. Therefore, the functioning of the epsilon lycopene cyclase of the invention was detected by a change in the color of E. coli cultures that accumulate lycopene.

[0058] Another method of the present invention for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic ε-cyclase enzyme, comprises

[0059] a) providing a vector containing a polynucleotide encoding the full-length enzyme and a primer for hybridizing to the polynucleotide;

[0060] b) performing site-directed mutagenesis using the primer and the vector for obtaining a construct containing a truncated polynucleotide encoding a fragment of the enzyme;

[0061] c) transfecting a host cell with the construct and growing the host cell under conditions for expressing a truncated protein encoded by the truncated polynucleotide;

[0062] d) allowing enzyme catalysis with the truncated protein on an enzyme-specific substrate in the host cell,

[0063] wherein the substrate is preferably, lycopene or another symmetrical substrate of the pathway of carotenoid synthesis and metabolism; and

[0064] e) identifying the enzyme-catalyzing domain encoded by the truncated protein by formation of a carotenoid compound from the enzyme catalysis of step d).

[0065] Another method of the present invention for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprises

[0066] a) providing a vector containing a polynucleotide encoding the full-length enzyme and a primer for hybridizing to the polynucleotide;

[0067] b) performing site-directed mutagenesis using the vector and the primer for obtaining a construct containing a site-directed mutant of the polynucleotide encoding the enzyme;

[0068] c) transfecting a host cell with the construct and growing the host cell under conditions for expressing a site-directed mutant of a protein encoded by the site-directed mutant of the polynucleotide;

[0069] d) allowing enzyme catalysis of the site-directed mutant of the protein on an enzyme-specific substrate in the host cell,

[0070] wherein the substrate is preferably, lycopene; and

[0071] e) identifying the enzyme-catalyzing domain encoded by the site-directed mutant of the protein by formation of a carotenoid compound from the enzyme catalysis of step d).

[0072] Primers used for obtaining truncated polynucleotide constructs or constructs containing site-directed mutants for polynucleotides encoding the enzymes of the invention include those described herein below. Preferred primers include but are not limited to SEQ ID NOS: 22-40.

[0073] The above-described methods were used to identify sequence determinants regulating catalytic activity in the preferred enzymes encoded by the polynucleotides of the invention.

[0074] More preferably, these methods were used to identify an internal region of six amino acid residues (underlined in the alignment of FIG. 4) regulating ring number determination for the preferred enzymes of the invention. In a preferred embodiment, the six amino acid segment implicated in determination of ring number for the lettuce and Arabidopsis LCYe is displayed in FIG. 6.

[0075] With respect to each of the 6 amino acid residues for the catalytic domain of the ε-cyclase enzyme family,

[0076] the first amino acid position of the 6 amino acids can be alanine (A), serine (S), glutamic acid (E) or asparagine (D), and is preferably S for an enzyme introducing two epsilon rings;

[0077] the second amino acid position of the 6 amino acid region can be arginine (R), leucine (L), histidine (H) or isoleucine (1), and is preferably H or R for an enzyme introducing two rings;

[0078] the third amino acid position of the 6 amino acid region can be isoleucine (I) or leucine (L), and is preferably I for an enzyme introducing two rings;

[0079] the fourth amino acid position of the 6 amino acid region can be valine (V) or leucine (L), and is preferably V for an enzyme introducing two rings;

[0080] the fifth amino acid position of the 6 amino acid region can be glutamine (Q), leucine (L) or lysine (K), and is preferably L for an enzyme introducing two rings; and

[0081] the sixth amino acid position of the 6 amino acid region can be phenylalanine (F), leucine (L), methionine (M) or leucine (L), and is preferably M for an enzyme introducing two rings.

[0082] The amino acid residues for the catalytic region of the lettuce ε-cyclase (lettuce LCYe) producing ε,ψ-carotene are SHIVLM (SEQ ID NO: 41) or SRIVLM (SEQ ID NO: 42).

[0083] The amino acid residues for the catalytic region of the Arabidopsis 6-cyclase (Arabidopsis LCYe) producing ε,ψ-carotene are ALIVQF (SEQ ID NO: 43).

[0084] The amino acid residues for the catalytic region of the potato ε-cyclase (potato LCYe) producing ε,ψ-carotene are ALILQL (SEQ ID NO: 44).

[0085] The amino acid residues for the catalytic region of the tomato ε-cyclase (tomato LCYe) producing ε,ψ-carotene are ALILQL (SEQ ID NO: 45).

[0086] The amino acid residues for the catalytic region of the marigold ε-cyclase (marigold LCYe) producing ε,ψ-carotene are ALIVQM (SEQ ID NO: 46).

[0087] The amino acid residues for the catalytic region of the Adonis ε-cyclase (Adonis LCYe1) producing ε,ψ- and ε,ε-carotene are ELIVQL (SEQ ID NO: 47).

[0088] Finally, the amino acid residues for the catalytic region of the Arabidopsis β-cyclase (Arabidopsis LCYb) producing β,β-carotene are DILLKL (SEQ ID NO: 48).

[0089] These methods were use to identify a single amino acid that determines whether the monocyclic δ-carotene or the bicyclic ε-carotene is produced from lycopene through the action of the Arabidopsis and lettuce ε-cyclases. More preferably, for most efficient synthesis of two epsilon rings, Arabidopsis or other monocyclic epsilon cyclase catalytic domains would be entirely replaced with a catalytic domain of lettuce comprising SHIVLM (SEQ ID NO: 41) or SRIVLM (SEQ ID NO: 42). Polynucleotides containing site-directed mutations in the region encoding the catalytic domain for Arabidopsis F-cyclase (LCYe) were constructed by replacing single amino acid residues for each of the naturally occurring residues of this enzyme. The inventors determined that conversion of lycopene into a bicyclic ε-carotene is primarily dependent on the second amino acid position of the 6 amino acid catalytic region of the Arabidopsis and lettuce ε-cyclase enzyme being H or R.

[0090] The relatedness of two carotenoid cyclase enzymes of differing functionality has allowed the Inventors to identify an amino acid residue that determines the distinctive properties of each. The success of this discovery demonstrates the utility of a domain swapping approach for identification of regions and residues of importance in the catalytic properties of a low abundance, membrane-associated enzyme (see also 29, 30 for use of a similar approach for soluble enzymes). This approach holds promise for identifying regions and residues integral to the functioning of other members of the carotenoid cyclase family (FIG. 7).

[0091] Transgenic organisms can be constructed which include the polynucleotides of the invention by methods described in PCT/US99/10461, and herein incorporated by reference in its entirety. The incorporation of chimeric polynucleotides containing the catalytic domain of one gene from one species inserted into the analogous sequence of a related gene from another species, can allow the controlling of carotenoid synthesis, content or composition in the host cell. In another approach, polynucleotides containing site-directed mutations in the catalytic domain of the ε-cyclase gene can also be transfected into host cells. More preferably, organisms which do not otherwise produce ε,ε-carotene such as Arabidopsis, can be transfected with the lettuce enzyme, a chimeric enzyme containing the lettuce catalytic domain or a modified form of the endogenous epsilon cyclase gene. Alternatively, mutations in the catalytic domain of the endogenous gene may be introduced and selected.

[0092] Any of the chimeric constructs of the invention can be used to increase the amount of bicyclic ε-carotene in an organism and thereby alter the nutritional value, pharmacology or visual appearance of the organism. In addition, the transformed organism can be used in the formulation of therapeutic agents, for example treatment of cancer (32, 33).

[0093] Appropriate transgenic hosts include plants such as marigold, tomato, pepper, banana, potato, and the like. Essentially any plant is suitable for expressing the preferred chimeric enzyme constructs, but the preferred plants are those which already produce high levels of carotenoids, and those which are normally ingested as foods or used as a source of carotenoid pigments. In particular, plants which bear fruit can be genetically manipulated to provide tissue-specific expression in fruit. Marigold is a particularly preferred host, because it can be used as a “bioreactor” for bulk production of carotenoids, and is actually grown commercially as a carotenoid source for chicken feed. For expression in marigold, a promoter can be used which is flower-specific. Another preferred transgenic plant is tomato, because this plant already produces high levels of lycopene, and it has been reported that there is a correlation between consuming tomatos and decreased incidence of colon cancer. Also, preferred are plants wherein the visual appearance is characterized by accumulation of carotenoids wherein the color properties could be changed by introduction of a modified epsilon cyclase.

[0094] The objects of the invention have been achieved by a series experiments, some of which are described by way of the following non-limiting examples.

EXAMPLES Example 1 Plant ε-Cyclase cDNAs

[0095] A Lactuca safiva var. romaine (romaine lettuce) leaf cDNA library was obtained from Dr. Harry Yamamoto (12). An Adonis aestivalis var. palaestina flower cDNA library has been described (13). The libraries were screened for cDNAs encoding lycopene cyclases by functional “color” complementation in Escherichia coli as previously described (7). A Solanum tuberosum (potato) ε-cyclase cDNA (GenBank accession 827545) was obtained from Dr. Nicholas J. Provart, Institut fuer Genbiologische Forschung, Berlin, Germany. Nucleotide sequences of the various cDNAs, and of chimera and mutants of these (see below), were determined by the DNA Sequencing Facility of the Center for Agricultural Biotechnology at the University of Maryland.

Example 2 Construction and Characterization of Chimeric and Truncated ε-Cyclases, and Site-Directed Mutants Thereof

[0096] A. Synthesis of Chimera by Inverse PCR

[0097] An inverse PCR strategy (FIG. 2) was developed to create chimera of the lettuce and Arabidopsis E-cyclase cDNAs. Plasmids containing both cDNAs, in tandem and in the same orientation, were first constructed. Plasmid templates were linearized by digestion with an appropriate restriction enzyme (BIpI for plasmid pLse/Ate, see FIG. 2; NcoI for pAte/Lse). PCR was performed in 100 μl tubes in an MJ Research PTC-150-25 MiniCycler with heated lid. The reaction volume of 50 μl contained 100 ng of template DNA and 2.5 units of Platinum Pfx DNA polymerase (Life Technologies). Concentrations of primers, dNTPs, magnesium, and buffer components were as suggested in the manufacturer's protocol. Oligonucleotide primer combinations used to construct the various chimera are listed in Table 1. TABLE 1 Oligonucleotides for construction of chimeric and truncated ε-cyclases and site- directed mutants Primer Name and Construct Junction *Primer Sequence Lse/Ste 262/108 (none) ELGG/PRVS conserved AvaII sites: GGWCC (SEQ ID NO: 1) Lse/Ste 285/6 (none) YDPD/LGLQ conserved BglII sites: AGATCT (SEQ ID NO: 2) Lse/Ate 323/320 Lse323N KIFF/EETC cgaagaatatttttgttggagaca (SEQ ID NO: 3) Ate324C aggagacatgtttggcctcaa (SEQ ID NO: 4) Lse/Ate 443/435 Lse443N WPLE/RKRQ ttcaagtggccacaatgtttc (SEQ ID NO: 5) Ate444C aggaaaagacagagagcattctt (SEQ ID NO: 6) Lse/Ate 461/453 Lse461N IVLM/DTEG attagcacgatgtgtgatagtcc (SEQ ID NO: 7) Ate462C ggataccgaaggcattagaag (SEQ ID NO: 8) Lse/Ate 498/490 Lse498N IIFA/LYMF gcaaatattatcaaatccgttgaaga (SEQ ID NO: 9) Ate499C tttatacatgttcgtcatttcaccaaa (SEQ ID NO: 10) Ate/Lse 390/395 Ate394N ATGY/SVVR tagcctgttgcgggatgta (SEQ ID NO: 11) Lse395C ttcagttgttcgatctttgtcag (SEQ ID NO: 12) Ate/Lse 408/413 AteN412 VIAE/ILRQ tctgcgatgactgatgcatatt (SEQ ID NO: 13) LseC314 (typo by gattttaagacaagatcaatctaaagagatg (SEQ ID NO: 14) vendor) Ate/Lse 420/430 AteN429 QINS/NISK ctgttgatctgtttggtagtctcttc (SEQ ID NO: 15) LseC430 taacatttcaaaacaagcatgggaaa (SEQ ID NO: 16) Ate/Lse 440/450 AteN449 RQRA/FFLF atgctctctgtcttttcctttctg (SEQ ID NO: 17) LseC450 tctttctattcggactatcacacatc (SEQ ID NO: 18) Ate/Lse 455/465 AteN464 FDTE/GTRT cttcggtatcgaattgaactatgag (SEQ ID NO: 19) LseC465 gaacacgtacatttttccgtactttc (SEQ ID NO: 20) Ate (none) Full length cDNA in pBluescript SK-; not a (SEQ ID NO: 21) LacZ fusion Ate Δ11N EPS-deltall caAcCatggcggtttcaacatttcc (SEQ ID NO: 22) (NcoI) Ate Δ58N EPS-DETTA59 tgtg CCATggtgagagaagatttcgctgac (SEQ ID NO: 23) (NcoI) Ate Δ82N EPS-delta82 ttc CCatgG agcagaacaaagatatggatga (SEQ ID NO: 24) (NcoI) Ate Δ88N EPS-delta88 aag CCatggatgaacagtctaagcttgttg (SEQ ID NO: 25) (NcoI) Ate Δ103N (none) Used MunI/mung bean + fused to NcoI/Klenow (SEQ ID NO: 26) of pTrcHisA Ate Δ23C Ate/K511tag gatgattgatgagaccttA tctcaaattgtttggtg (SEQ ID NO: 27) (=taa) Ate ALIVQF447- Ate/456-61 SHIVLM gccttcggtatcCaTtAg CacGatgTgtgAaagaccaaagag (SEQ ID NO: 28) 52/SHIVLM (DraIII) Ate L448H AteL457H/FspI ggtatcgaattgaactatg TgCgcaagaccaaagagaaag (SEQ ID NO: 29) Ate L448D AteL457D/EcoRV ggtatcgaattgaac GatATC tgcaagaccaaagagaaag (SEQ ID NO: 30) Ate L448R AteL457R/BssHII ggtatcgaattgaactatgCgCgcaagaccaaagagaaag (SEQ ID NO: 31) Ate A447D AteA456D/BglII cgaattgaactatgagATcT agaccaaagagaaagaatgc (SEQ ID NO: 32) Lse (none) Full length cDNA in pBluescript SK-; not (SEQ ID NO: 33) a lacZ fusion Lse Δ92N (none) Used pre-existing NcoI; fusion in TrcHisA (SEQ ID NO: 34) Lse Δ107N (none) Used MunI/mung bean + fused to NcoI/Klenow (SEQ ID NO: 35) of pTrcHisA Lse Δ23C Lse/M511tag atgtctaaccagttccTA tctcaagctgtgaggtgc (SEQ ID NO: 36) Lse H457L Lse/H457L ccctctagatccattagcacgatgAgtgatagtccgaatagaaag (SEQ ID NO: 37) Lse H457D LseH457D/EcoRV cctctagatccattagcacgatAtC tgatagtccgaatagaaag (SEQ ID NO: 38) Lse H457R Lse/H457R/XhoI ctagatccattagcacgatT CTCgaG agtccgaatagaaagaagg (SEQ ID NO: 39) Lse L460Q Lse/L460Q gttccctctagatccattTgcacgatgtgtgatag (SEQ ID NO: 40)

[0098] Typical cycling parameters were: 94° C. for 3 min, fifteen cycles of 94/55/68° C. for 20/60/360 sec. ten cycles of 94/55/68° C. for 20/60/360+15 additional sec each cycle, 68° C. for 10 min, and hold at 4° C. PCR products were purified by gel electrophoresis (0.8% SeaKem GTG agarose; FMC BioProducts) and recovered from gels using the GENECLEAN kit (Bio101, Inc.). The ends of the recovered PCR products were phosphorylated with T4 polynucleotide kinase (New England BioLabs, Inc.; 5 units of enzyme and one-half of the recovered PCR product in a final volume of 10 μl) with incubation at 37° C. for 30 min, and the reactions were then cooled on ice. T4 DNA ligase (0.5 μl containing 200 NEB units; New England BioLabs) was then added, and samples were incubated for 12-16 h at 15° C. One μl of each ligation mixture was used to transform chemically competent E. coli (25 μl of XL10 Gold cells; Stratagene Cloning Systems, Inc.), and the transformation mixture was plated on a single large (15 cm) Luria-Bertani (LB) agar (1.5%, w/v) plate containing 150 μg/ml ampicillin (sodium salt). The resulting colonies (typically several thousand) were collected and combined in 5-10 ml LB medium. The plasmids were purified and transformed into a pink colored, lycopene-accumulating E. coli strain (14) for analysis. Pigments were extracted and analyzed from several of the resulting yellow colonies (i.e., a yellow color is indicative of an active cyclase). Usually more than 50% of the colonies were yellow. The plasmid from one of the colonies was recovered, and the nucleotide sequence was determined to verify the construct.

[0099] An AvalI site located in the same relative position in the lettuce and potato ε-cyclase cDNAs was used to construct a chimeric lettuce/potato ε-cyclase in which the first 262 amino acids of the encoded polypeptide derived from the lettuce cDNA and the subsequent 272 were specified by the potato cDNA. The plasmid containing this chimeric cDNA is referred to as pLse262/Ste108. The product of this chimeric cDNA converted lycopene to δ-carotene in E. coli (data not shown) thereby indicating that the potato enzyme is a mono-ε-cyclase.

[0100] B. Synthesis of Truncations for and Site-Directed Mutants of ε-Cyclases by Site-Directed Mutagenesis.

[0101] N-terminal truncations of cDNAs encoding the Arabidopsis and lettuce lycopene ε-cyclases were created using restriction sites or by using the CHAMELEON™ Double-Stranded, Site-Directed Mutagenesis Kit (Stratagene Cloning systems, Inc.) to introduce a NcoI site at the position desired for the initiation codon. The resulting product was excised and inserted into the NcoI site of plasmid vector pTrcHisA (Invitrogen, Inc.), downstream of and in frame with the inducible Trc promoter. C-terminal truncations were created by introducing termination codons at the desired positions. Various other site-specific mutations, usually accompanied by the introduction of a restriction site to facilitate the identification of mutants, were also created with the CHAMELEON™ kit. Primers (SEQ ID NOS: 22-40) used to introduce the various mutations are listed in Table 1. Mutations were confirmed by analysis of the nucleotide sequence.

Example 3 Mapping and Identification of Catalytic Domains by Analysis of Chimera and Truncations for ε-Cyclase

[0102] Carotenoids with two ε-rings are uncommon in plants. A notable exception is romaine lettuce, where lactucaxanthin (ε,ε-carotene-3,3′-diol) comprises as much as 21% (mol/mol) of the total carotenoid pigment in the leaves (10, 11). The inventors selected twenty-six prospective lycopene ε-cyclase cDNAs in a screen of a romaine lettuce leaf cDNA library. Other than in length, the cDNAs appeared to be identical. The complete nucleotide sequence of the longest cDNA was ascertained, and a plasmid construct, pDY4 containing a subcloned cDNA, was introduced into a lycopene-accumulating strain of E. coli for analysis of the activity of the encoded enzyme.

[0103] Accumulation of carotenoids with two ε rings in lettuce is known to occur in vivo (10, 11). Introduction of the lettuce ε-cyclase cDNA into the lycopene-producing E. coli yielded predominantly (>90%) ε-carotene as indicated by the HPLC retention time (FIG. 3, panel A) and absorption spectrum (FIG. 3, panel D) of the major product. In marked contrast, introduction of the Arabidopsis LCYe into lycopene-accumulating E. coli yielded, as earlier reported (7), approximately 98% of monocyclic δ-carotene for the total amount of carotenoid produced (FIG. 3, panel B; absorption spectrum in panel E). The HPLC elution profile of a lycopene-accumulating control culture (FIG. 3, panel C), and the absorption spectrum of the major compound in this strain (lycopene; FIG. 3, panel F) are also displayed for comparison. Elution times were ca. 12.2 min for lycopase (ψ,ψ-carotene), 14.3 min for δ-carotene (ε,ψ-carotene), and 17.1 min for ε-carotene (ε,ε-carotene).

[0104] The Arabidopsis and lettuce lycopene ε-cyclases are substantially similar in their deduced amino acid sequences (ca. 77% overall identity; FIG. 4 (the alignment was created using Clustal X version 1.8 (17). GenBank accession numbers are listed at the ends of the sequences. Asterisks above the alignment are spaced every 10 residues. Numbers to the right denote the number of the amino acid residue that ends the row)) and closely resemble other known LCYe (FIG. 8).

[0105] Specific amino acid differences at the N-terminus of the ε-cyclases were initially thought to be involved in determination of ring number. The lettuce and Arabidopsis ε-cyclase cDNAs were therefore modified so as to produce polypeptides truncated at the N terminus. Carotenoids that accumulate in an E. coli strain containing the indicated cDNA subcloned into plasmid vector pBluescript SK-, and that otherwise accumulates only lycopene (ψ,ψ-carotene), are indicated to the right. Only the predominant carotenoid (>90% of the total in all cases) is listed. Solid black vertical lines connecting the Arabidopsis and lettuce cyclases at the top of the figure indicate identically-conserved amino acid residues. LsE/AtE 323/320 defines a chimera consisting of the 5′ portion of the lettuce ε-cyclase cDNA up to and including nucleotide bases specifying amino acid residues 323 and the 3′ portion of the Arabidopsis ε-cyclase cDNA beginning with nucleotide bases that encode amino acid residue 320 and proceeding to the end of the cDNA.

[0106] Substantial portions of the N-termini of the lettuce and Arabidopsis ε-cyclases were found to be nonessential to catalytic function (FIG. 5), but all truncations that yielded an active enzyme did not alter the mixture of products produced from lycopene in E. coli (FIG. 5).

[0107] C-terminal truncations were also constructed, and for those truncations where even a relatively small portion of the polypeptide was deleted, ε-cyclase enzyme activity in E. coli was completely eliminated (FIG. 5).

[0108] These catalytic regions were further characterized using a series of chimeric cDNAs encoding portions of both the lettuce and Arabidopsis LCYe. The chimeric cDNAs were constructed by an inverse PCR-based method (FIG. 2) to minimize constraints on the choice of the chimera junction. Construction of the chimeras, and the activity of their respective polypeptide products are presented in FIG. 5. Constructs for the lettuce bi-ε-cyclase with the N-terminal portion and the Arabidopsis bi-ε-cyclase for the C-terminal portion of the chimeric cDNAs are shown. Characterization of these chimeric lettuce/Arabidopsis F-cyclases defined a region of six amino acids (underlined in the alignment of FIG. 4) that is involved in ring number determination. These initial experiments did not rule out that other amino acids elsewhere in the polypeptides might also influence the ring number. A second series of chimeras using the Arabidopsis cDNA as the N-terminus and the lettuce cDNA as the C-terminus, identified the same 6 amino acid domain as conferring ring number determination (FIG. 5).

[0109] Amino acids within this region and in the context of the rest of the amino acid sequence are able to confer whether the enzyme adds one or two rings to lycopene. The six amino acid segment implicated in determination of ring number is displayed in FIG. 6 for the lettuce and Arabidopsis LCYe.

[0110] Similarly conserved residues are shown in black text on a gray background. Three amino acid residues in the lettuce bi-ε-cyclase that differ significantly from those in the known mono-ε-cyclases are in white text on a black background. Sequences of an Arabidopsis LCYb (a bicyclase) and an Adonis LCYe of mixed function are displayed below the lettuce LCYe with two residues of interest shown in white text on a black background. Similarity was defined according to the Blosum 45 scoring matrix (19): DE, NH, ST, QKR, FYW, LIVM). GenBank accession numbers: Adonis LCYe1, AF321535; Arabidopsis LCYb, U50739; Arabidopsis LCYe, U50738; lettuce LCYe, AF321538; marigold LCYe, AF251016; potato LCYe, AF321537; tomato LCYe, Y14387.

[0111] For comparison, sequences in this region for other known mono-ε-cyclases are also displayed. The nucleotides that specify these amino acids in the Arabidopsis ε-cyclase (ALIVQF) were replaced with those that specify the amino acids of the lettuce ε-cyclase (SHIVLM). The enzyme produced by this cDNA (mutant AtE ALIVQF447-52SHIVLM) functions even better than the lettuce ε-cyclase (Table 2), confirming that determination of ring number is influenced by one or more of the amino acids in this small region of the polypeptide.

[0112] Within the six amino acid region mapped by the chimeric lettuce and Arabidopsis E-cyclases (FIG. 5), only four residues differ between the two sequences (FIG. 6). Of these four differences, the residue at position M461 of lettuce vs. the residue at position F452 of Arabidopsis is likely unimportant because it is a conservative replacement and because the marigold mono-ε-cyclase (18) also has an M residue in this position (FIG. 6). The residue in position H457 of lettuce (vs. residue in position L448 in Arabidopsis) and the residue in position L460 of lettuce (vs. residue in position Q451 in Arabidopsis) are the most conspicuous differences relative to the sequence of the Arabidopsis LCYe.

[0113] The inventors have shown that the change of a single amino acid in the polypeptide sequences of the Arabidopsis and lettuce lycopene ε-cyclases has a profound influence on the ability of these enzymes to add a second ε-ring to the symmetrical substrate lycopene. The gain of function engendered in the Arabidopsis LCYe mutants L448H and L448R and the importance of this specific amino acid residue are all the more compelling when contrasted against the loss of function in the lettuce LCYe mutant H457L. This single amino acid at position 448, thus regulates molecular switching for ring number determination by lycopene ε-cyclases.

[0114] The lycopene ε-cyclases are members of an extended family of carotenoid modifying enzymes (FIG. 7) that includes capsanthin-capsorubin synthase (CCS; 21) and the recently identified neoxanthin synthase (NSY; 22, 23), as well as lycopene P-cyclase (LCYb; 7, 18, 20, 26).

[0115] A lycopene β-cyclase from the cyanobacterium Synechococcus PCC7942 (14) was used as the outgroup. Branch lengths are drawn to scale. Bootstrap values greater than 50% for 10,000 replicates with a seed value of 111 are indicated. The analysis encompassed 398 positions, beginning with the initiating Met of the Synechococcus cyclase, and excluded those positions with gaps in the alignment. The amino acid sequence alignment and GenBank accession numbers for the nucleotide sequences are shown in FIG. 8. The method of Saitou and Nei (24) was used to construct the tree. Distances were corrected for multiple substitutions (25).

[0116] LCYb and CCS each act at both ends of their respective symmetrical substrates, while NSY acts at only one end of the symmetrical violaxanthin. The known plant LCYb do not contain a basic residue in the position corresponding to H457 of the lettuce LCYe; instead they contain the nonpolar I residue (FIGS. 6 and 8). The ability of LCYb to add two β rings to lycopene must, therefore, derive from an alternative solution to that which confers a bicyclase activity to the lettuce LCYe.

[0117] A more complete conversion to ε-carotene by Arabidopsis LCYe mutant ALIVQF44742SHIVLM (98% ε-carotene) was observed compared to the mutant L448H (92% ε-carotene; Table 2). This indicated that the preceding amino acid residue (a nonpolar A447 in Arabidopsis vs. a polar S456 of lettuce) influences ring number determination. The known plant β-cyclases contain an acidic residue (D) in this position. Two closely-related Adonis aestivalis ε-cyclase cDNAs (FIG. 6) also specify an acidic amino acid (E) in this position, but do not otherwise differ significantly from mono-ε-cyclases in this region (FIG. 6). The Adonis LCYe produce a preponderance of ε-carotene in lycopene-accumulating E. coli (Table 2). However, conversion of the A447 residue of the Arabidopsis LCYe to a D did not yield a bicyclase (Table 2), indicating that the identity of this residue does not, by itself, determine ring number.

[0118] Lycopene is a symmetrical, nonpolar C₄₀ hydrocarbon (FIG. 1) that is insoluble in aqueous solutions and accumulates in membranes and oil bodies of plant cells. There is considerable uncertainty regarding the orientation of lycopene and other carotenoids in the plane of the membrane (see 27 for a discussion), and also of the position of the cyclase enzyme within or on the surface of the membrane (7, 26). If lycopene spans (i.e. is perpendicular to the plane of the membrane) or partially penetrates the membrane, then the two ends of the molecule will almost certainly not be equally accessible to the cyclase. There is experimental evidence that the two ends of β-carotene differ in accessibility to the hydroxylase enzyme that converts this compound to zeaxanthin (β,β-carotene-3,3′-diol; 28). As was suggested for the hydoxylase enzyme (26, 28), the addition of two rings to lycopene may depend on an ability of the cyclase to form dimers, whereby binding of the more accessible end of the substrate by one of the subunits would serve to bring the other end of the carotenoid molecule into proximity of the cognate subunit where catalysis could then proceed. The region encompassing the L447 residue of the Arabidopsis LCYe might then constitute an interfacial surface that mediates subunit interaction.

Example 4 Identification of Amino Acid Residues Conferring ε-Cyclase Activity

[0119] Plasmids containing individual ε-cyclase cDNAs, chimera or site-directed mutants were transformed into lycopene-accumulating E. coli strain TOP10 (14). Cultures in six ml LB medium containing 150 μg/ml ampicillin and 30 μg/ml chloramphenicol were grown for 1 day with shaking in darkness at 28° C. as described previously (15). Cells were harvested by centrifugation and pigments were extracted and analyzed by HPLC essentially as described previously (7, 16), except that an isocratic mobile phase of 40% B was used for the analysis. Pigments were identified on the basis of absorption spectra and HPLC retention times relative to standard compounds. TABLE 2 Activity of lycopene ε-cyclases and site-directed mutants with lycopene (ψ,ψ-carotene) as substrate in E. coli *Carotenoids ε-Cyclase cDNA Mutation lyc:del:eps (none) — 100:0:0 Arabidopsis (AtE) wild type (y2)  1:98:1 AtE ALIVQF447-52SHIVLM  0:2:98 AtE L448H  0:8:92 AtE L448R  0:8:92 AtE L448D  37:56:8 AtE A447D  1:98:1 Lettuce (LsE) wild type (DY4)  3:8:90 LsE H457R  3:6:91 LsE H457D  22:18:60 LsE H457L  17:73:10 Adonis (AaE1) wild type (Ad3)  0:44:56

[0120] The activity of a lettuce L460Q mutant did not differ significantly from that provided by the wild type lettuce cDNA. The lettuce H457L mutant, in contrast, exhibited an activity comparable to that of the Arabidopsis enzyme: δ-carotene was the predominant product accumulated in E. coli. Conversely, the corresponding Arabidopsis mutant, L448H, gained the ability to produce ε-carotene as the predominant product. Thus, the identity of the amino acid residue within this single position of the lettuce and Arabidopsis sequences specifies whether a monocyclase or bicyclase activity results.

[0121] The Arabidopsis L448 and lettuce H457 were also changed to D and R residues in order to gain insight as to what properties of the residue in this position influence the determination of ring number. For both the Arabidopsis and lettuce ε-cyclases, conversion to an R, like H a positively charged residue, gave results essentially identical to those obtained with an H codon at this position (see AtE L448R and LsE H457R). Conversion to D (AtE L447D and LsE H457D), a negatively charged residue, greatly impaired the overall activity of the enzymes (i.e. a substantial proportion of the substrate lycopene remained) and reduced, though did not eliminate, formation of ε-carotene.

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1 97 1 5 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 1 ggwcc 5 2 6 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 2 agatct 6 3 24 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 3 cgaagaatat ttttgttgga gaca 24 4 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 4 aggagacatg tttggcctca a 21 5 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 5 ttcaagtggc cacaatgttt c 21 6 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 6 aggaaaagac agagagcatt ctt 23 7 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 7 attagcacga tgtgtgatag tcc 23 8 21 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 8 ggataccgaa ggcattagaa g 21 9 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 9 gcaaatatta tcaaatccgt tgaaga 26 10 27 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 10 tttatacatg ttcgtcattt caccaaa 27 11 19 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 11 tagcctgttg cgggatgta 19 12 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 12 ttcagttgtt cgatctttgt cag 23 13 22 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 13 tctgcgatga ctgatgcata tt 22 14 31 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 14 gattttaaga caagatcaat ctaaagagat g 31 15 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 15 ctgttgatct gtttggtagt ctcttc 26 16 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 16 taacatttca aaacaagcat gggaaa 26 17 24 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 17 atgctctctg tcttttcctt tctg 24 18 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 18 tctttctatt cggactatca cacatc 26 19 25 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 19 cttcggtatc gaattgaact atgag 25 20 26 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 20 gaacacgtac atttttccgt actttc 26 21 1860 DNA Arabidopsis thaliana 21 acaaaaggaa ataattagat tcctctttct gcttgctata ccttgataga acaatataac 60 aatggtgtaa gtcttctcgc tgtattcgaa attatttgga ggaggaaaat ggagtgtgtt 120 ggggctagga atttcgcagc aatggcggtt tcaacatttc cgtcatggag ttgtcgaagg 180 aaatttccag tggttaagag atacagctat aggaatattc gtttcggttt gtgtagtgtc 240 agagctagcg gcggcggaag ttccggtagt gagagttgtg tagcggtgag agaagatttc 300 gctgacgaag aagattttgt gaaagctggt ggttctgaga ttctatttgt tcaaatgcag 360 cagaacaaag atatggatga acagtctaag cttgttgata agttgcctcc tatatcaatt 420 ggtgatggtg ctttggatca tgtggttatt ggttgtggtc ctgctggttt agccttggct 480 gcagaatcag ctaagcttgg attaaaagtt ggactcattg gtccagatct tccttttact 540 aacaattacg gtgtttggga agatgaattc aatgatcttg ggctgcaaaa atgtattgag 600 catgtttgga gagagactat tgtgtatctg gatgatgaca agcctattac cattggccgt 660 gcttatggaa gagttagtcg acgtttgctc catgaggagc ttttgaggag gtgtgtcgag 720 tcaggtgtct cgtaccttag ctcgaaagtt gacagcataa cagaagcttc tgatggcctt 780 agacttgttg cttgtgacga caataacgtc attccctgca ggcttgccac tgttgcttct 840 ggagcagctt cgggaaagct cttgcaatac gaagttggtg gacctagagt ctgtgtgcaa 900 actgcatacg gcgtggaggt tgaggtggaa aatagtccat atgatccaga tcaaatggtt 960 ttcatggatt acagagatta tactaacgag aaagttcgga gcttagaagc tgagtatcca 1020 acgtttctgt acgccatgcc tatgacaaag tcaagactct tcttcgagga gacatgtttg 1080 gcctcaaaag atgtcatgcc ctttgatttg ctaaaaacga agctcatgtt aagattagat 1140 acactcggaa ttcgaattct aaagacttac gaagaggagt ggtcctatat cccagttggt 1200 ggttccttgc caaacaccga acaaaagaat ctcgcctttg gtgctgccgc tagcatggta 1260 catcccgcaa caggctattc agttgtgaga tctttgtctg aagctccaaa atatgcatca 1320 gtcatcgcag agatactaag agaagagact accaaacaga tcaacagtaa tatttcaaga 1380 caagcttggg atactttatg gccaccagaa aggaaaagac agagagcatt ctttctcttt 1440 ggtcttgcac tcatagttca attcgatacc gaaggcatta gaagcttctt ccgtactttc 1500 ttccgccttc caaaatggat gtggcaaggg tttctaggat caacattaac atcaggagat 1560 ctcgttctct ttgctttata catgttcgtc atttcaccaa acaatttgag aaaaggtctc 1620 atcaatcatc tcatctctga tccaaccgga gcaaccatga taaaaaccta tctcaaagta 1680 tgatttactt atcaactctt aggtttgtgt atatatatgt tgatttatct gaataatcga 1740 tcaaagaatg gtatgtgggt tactaggaag ttggaaacaa acatgtatag aatctaagga 1800 gtgatcgaaa tggagatgga aacgaaaaga aaaaaatcag tctttgtttt gtggttagtg 1860 22 25 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 22 caaccatggc ggtttcaaca tttcc 25 23 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 23 tgtgccatgg tgagagaaga tttcgctgac 30 24 31 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 24 ttcccatgga gcagaacaaa gatatggatg a 31 25 30 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 25 aagccatgga tgaacagtct aagcttgttg 30 26 6 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 26 ccatgg 6 27 36 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 27 gatgattgat gagaccttat ctcaaattgt ttggtg 36 28 42 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 28 gccttcggta tccattagca cgatgtgtga aagaccaaag ag 42 29 40 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 29 ggtatcgaat tgaactatgt gcgcaagacc aaagagaaag 40 30 40 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 30 ggtatcgaat tgaacgatat ctgcaagacc aaagagaaag 40 31 40 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 31 ggtatcgaat tgaactatgc gcgcaagacc aaagagaaag 40 32 40 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 32 cgaattgaac tatgagatct agaccaaaga gaaagaatgc 40 33 1780 DNA Lactuca sativa 33 gaaacaaatg acgtgaaagt tcttcaaaat tgaattaatt gtaatcctga aaacttgatt 60 tgtgatagaa gaatcaatgg agtgctttgg agctcgaaac atgacggcaa caatggcggt 120 ttttacgtgc cctagattca cggactgtaa tatcaggcac aaattttcgt tactgaaaca 180 acgaagattt actaatttat cagcatcgtc ttcgttgcgt caaattaagt gcagcgctaa 240 aagcgaccgt tgtgtagtgg ataaacaagg gatttccgta gcagacgaag aagattatgt 300 gaaggccggt ggatcggagc tgttttttgt tcaaatgcag cggactaagt ccatggaaag 360 ccagtctaaa ctttccgaaa agctagcaca gataccaatt ggaaattgca tacttgatct 420 ggttgtaatc ggttgtggcc ctgctggcct tgctcttgct gcagagtcag ccaaactagg 480 gttgaacgtt ggactcattg gccctgatct tccttttaca aacaattatg gtgtttggca 540 ggatgaattt ataggtcttg gacttgaagg atgcattgaa cattcttgga aagatactct 600 tgtatacctt gatgatgctg atcccatccg cataggtcgt gcatatggca gagttcatcg 660 tgatttactt catgaagagt tgttaagaag gtgtgtggaa tcaggtgttt catatctaag 720 ctccaaagta gaaagaatca ctgaagctcc aaatggctat agtctcattg aatgtgaagg 780 caatatcacc attccatgca ggcttgctac tgttgcatca ggggcagctt cagggaaatt 840 tctggagtat gaacttgggg gtccccgtgt ttgtgtccaa acagcttatg gtatagaggt 900 tgaggttgaa aacaacccct atgatccaga tctaatggtg ttcatggatt atagagactt 960 ctcaaaacat aaaccggaat ctttagaagc aaaatatccg actttcctct atgtcatggc 1020 catgtctcca acaaaaatat tcttcgagga aacttgttta gcttcaagag aagccatgcc 1080 tttcaatctt ctaaagtcca aactcatgtc acgattaaag gcaatgggta tccgaataac 1140 aagaacgtac gaagaggaat ggtcgtatat ccccgtaggt ggatcgttac ctaatacaga 1200 acaaaagaat ctcgcatttg gtgctgcagc tagtatggtg caccctgcca cagggtattc 1260 agttgttcga tctttgtcag aagctcctaa ttatgcagca gtcattgcta agattttaag 1320 acaagatcaa tctaaagaga tgatttctct tggaaaatac actaacattt caaaacaagc 1380 atgggaaaca ttgtggccac ttgaaaggaa aagacagcga gccttctttc tattcggact 1440 atcacacatc gtgctaatgg atctagaggg aacacgtaca tttttccgta ctttctttcg 1500 tttgcccaaa tggatgtggt ggggattttt ggggtcttct ttatcttcaa cggatttgat 1560 aatatttgcg ctttatatgt ttgtgatagc acctcacagc ttgagaatgg aactggttag 1620 acatctactt tctgatccga caggggcaac tatggtaaaa gcatatctca ctatatagat 1680 ttagattata taaataatac ccatatcttg catatatata agccttattt atttcttttg 1740 tatccttaca acaacatact cgttaattat atgtttttta 1780 34 6 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 34 ccatgg 6 35 6 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 35 ccatgg 6 36 36 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 36 atgtctaacc agttcctatc tcaagctgtg aggtgc 36 37 45 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 37 ccctctagat ccattagcac gatgagtgat agtccgaata gaaag 45 38 44 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 38 cctctagatc cattagcacg atatctgata gtccgaatag aaag 44 39 45 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 39 ctagatccat tagcacgatt ctcgagagtc cgaatagaaa gaagg 45 40 35 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 40 gttccctcta gatccatttg cacgatgtgt gatag 35 41 6 PRT Lactuca sativa 41 Ser His Ile Val Leu Met 1 5 42 6 PRT Lactuca sativa 42 Ser Arg Ile Val Leu Met 1 5 43 6 PRT Arabidopsis sp. 43 Ala Leu Ile Val Gln Phe 1 5 44 6 PRT Solanum tuberosum 44 Ala Leu Ile Leu Gln Leu 1 5 45 6 PRT Lycopersicon esculentum 45 Ala Leu Ile Leu Gln Leu 1 5 46 6 PRT Tagetes erecta 46 Ala Leu Ile Val Gln Met 1 5 47 6 PRT Adonis aestivalis 47 Glu Leu Ile Val Gln Leu 1 5 48 6 PRT Arabidopsis sp. 48 Asp Ile Leu Leu Lys Leu 1 5 49 523 PRT Arabidopsis thaliana 49 Met Glu Cys Val Gly Ala Arg Asn Phe Ala Ala Met Ala Val Ser Thr 1 5 10 15 Phe Pro Ser Trp Ser Cys Arg Arg Lys Phe Pro Val Val Lys Arg Tyr 20 25 30 Ser Tyr Arg Asn Ile Arg Phe Gly Leu Cys Ser Val Arg Ala Ser Gly 35 40 45 Gly Gly Ser Ser Gly Ser Glu Ser Cys Val Ala Val Arg Glu Asp Phe 50 55 60 Ala Asp Glu Glu Asp Phe Val Lys Ala Gly Gly Ser Glu Ile Leu Phe 65 70 75 80 Val Gln Met Gln Gln Asn Lys Asp Met Asp Glu Gln Ser Lys Leu Val 85 90 95 Asp Lys Leu Pro Pro Ile Ser Ile Gly Asp Gly Ala Leu Asp His Val 100 105 110 Val Ile Gly Cys Gly Pro Ala Gly Leu Ala Leu Ala Ala Glu Ser Ala 115 120 125 Lys Leu Gly Leu Lys Val Gly Leu Ile Gly Pro Asp Leu Pro Phe Thr 130 135 140 Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Asn Asp Leu Gly Leu Gln 145 150 155 160 Lys Cys Ile Glu His Val Trp Arg Glu Thr Ile Val Tyr Leu Asp Asp 165 170 175 Asp Lys Pro Ile Thr Ile Gly Arg Ala Tyr Gly Arg Val Ser Arg Arg 180 185 190 Leu Leu His Glu Glu Leu Leu Arg Arg Cys Val Glu Ser Gly Val Ser 195 200 205 Tyr Leu Ser Ser Lys Val Asp Ser Ile Thr Glu Ala Ser Asp Gly Leu 210 215 220 Arg Leu Val Ala Cys Asp Asp Asn Asn Val Ile Pro Cys Arg Leu Ala 225 230 235 240 Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Leu Leu Gln Tyr Glu Val 245 250 255 Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr Gly Val Glu Val Glu 260 265 270 Val Glu Asn Ser Pro Tyr Asp Pro Asp Gln Met Val Phe Met Asp Tyr 275 280 285 Arg Asp Tyr Thr Asn Glu Lys Val Arg Ser Leu Glu Ala Glu Tyr Pro 290 295 300 Thr Phe Leu Tyr Ala Met Pro Met Thr Lys Ser Arg Leu Phe Phe Glu 305 310 315 320 Glu Thr Cys Leu Ala Ser Lys Asp Val Met Pro Phe Asp Leu Leu Lys 325 330 335 Thr Lys Leu Met Leu Arg Leu Asp Thr Leu Gly Ile Arg Ile Leu Lys 340 345 350 Thr Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val Gly Gly Ser Leu Pro 355 360 365 Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala Ala Ala Ser Met Val 370 375 380 His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser Leu Ser Glu Ala Pro 385 390 395 400 Lys Tyr Ala Ser Val Ile Ala Glu Ile Leu Arg Glu Glu Thr Thr Lys 405 410 415 Gln Ile Asn Ser Asn Ile Ser Arg Gln Ala Trp Asp Thr Leu Trp Pro 420 425 430 Pro Glu Arg Lys Arg Gln Arg Ala Phe Phe Leu Phe Gly Leu Ala Leu 435 440 445 Ile Val Gln Phe Asp Thr Glu Gly Ile Arg Ser Phe Phe Arg Thr Phe 450 455 460 Phe Arg Leu Pro Lys Trp Met Gln Gly Phe Leu Gly Ser Thr Leu Thr 465 470 475 480 Ser Gly Asp Leu Val Leu Phe Ala Leu Tyr Met Phe Val Ile Ser Pro 485 490 495 Asn Asn Leu Arg Lys Gly Leu Ile Asn His Leu Ile Ser Asp Pro Thr 500 505 510 Gly Ala Thr Met Ile Lys Thr Tyr Leu Lys Val 515 520 50 533 PRT Lactuca sativa 50 Met Glu Cys Phe Gly Ala Arg Asn Met Thr Ala Thr Met Ala Val Phe 1 5 10 15 Thr Cys Pro Arg Phe Thr Asp Cys Asn Ile Arg His Lys Phe Ser Leu 20 25 30 Leu Lys Gln Arg Arg Phe Thr Asn Leu Ser Ala Ser Ser Ser Leu Arg 35 40 45 Gln Ile Lys Cys Ser Ala Lys Ser Asp Arg Cys Val Val Asp Lys Gln 50 55 60 Gly Ile Ser Val Ala Asp Glu Glu Asp Tyr Val Lys Ala Gly Gly Ser 65 70 75 80 Glu Leu Phe Phe Val Gln Met Gln Arg Thr Lys Ser Met Glu Ser Gln 85 90 95 Ser Lys Leu Ser Glu Lys Leu Ala Gln Ile Pro Ile Gly Asn Cys Ile 100 105 110 Leu Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ala Leu Ala 115 120 125 Ala Glu Ser Ala Lys Leu Gly Leu Asn Val Gly Leu Ile Gly Pro Asp 130 135 140 Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Gln Asp Glu Phe Ile Gly 145 150 155 160 Leu Gly Leu Glu Gly Cys Ile Glu His Ser Trp Lys Asp Thr Leu Val 165 170 175 Tyr Leu Asp Asp Ala Asp Pro Ile Arg Ile Gly Arg Ala Tyr Gly Arg 180 185 190 Val His Arg Asp Leu Leu His Glu Glu Leu Leu Arg Arg Cys Val Glu 195 200 205 Ser Gly Val Ser Tyr Leu Ser Ser Lys Val Glu Arg Ile Thr Glu Ala 210 215 220 Pro Asn Gly Tyr Ser Leu Ile Glu Cys Glu Gly Asn Ile Thr Ile Pro 225 230 235 240 Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Phe Leu 245 250 255 Glu Tyr Glu Leu Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr Gly 260 265 270 Ile Glu Val Glu Val Glu Asn Asn Pro Tyr Asp Pro Asp Leu Met Val 275 280 285 Phe Met Asp Tyr Arg Asp Phe Ser Lys His Lys Pro Glu Ser Leu Glu 290 295 300 Ala Lys Tyr Pro Thr Phe Leu Tyr Val Met Ala Met Ser Pro Thr Lys 305 310 315 320 Ile Phe Phe Glu Glu Thr Cys Leu Ala Ser Arg Glu Ala Met Pro Phe 325 330 335 Asn Leu Leu Lys Ser Lys Leu Met Ser Arg Leu Lys Ala Met Gly Ile 340 345 350 Arg Ile Thr Arg Thr Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val Gly 355 360 365 Gly Ser Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala Ala 370 375 380 Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser Leu 385 390 395 400 Ser Glu Ala Pro Asn Tyr Ala Ala Val Ile Ala Lys Ile Leu Arg Gln 405 410 415 Asp Gln Ser Lys Glu Met Ile Ser Leu Gly Lys Tyr Thr Asn Ile Ser 420 425 430 Lys Gln Ala Trp Glu Thr Leu Trp Pro Leu Glu Arg Lys Arg Gln Arg 435 440 445 Ala Phe Phe Leu Phe Gly Leu Ser His Ile Val Leu Met Asp Leu Glu 450 455 460 Gly Thr Arg Thr Phe Phe Arg Thr Phe Phe Arg Leu Pro Lys Trp Met 465 470 475 480 Trp Trp Gly Phe Leu Gly Ser Ser Leu Ser Ser Thr Asp Leu Ile Ile 485 490 495 Phe Ala Leu Tyr Met Phe Val Ile Ala Pro His Ser Leu Arg Met Glu 500 505 510 Leu Val Arg His Leu Leu Ser Asp Pro Thr Gly Ala Thr Met Val Lys 515 520 525 Ala Tyr Leu Thr Ile 530 51 18 PRT Arabidopsis sp. 51 Phe Phe Leu Phe Gly Leu Ala Leu Ile Val Gln Phe Asp Thr Glu Gly 1 5 10 15 Ile Arg 52 18 PRT Solanum tuberosum 52 Phe Phe Leu Phe Gly Leu Ala Leu Ile Leu Gln Leu Asp Ile Glu Gly 1 5 10 15 Ile Arg 53 18 PRT Lycopersicon esculentum 53 Phe Phe Leu Phe Gly Leu Ala Leu Ile Leu Gln Leu Asp Ile Glu Gly 1 5 10 15 Ile Arg 54 18 PRT Tagetes erecta 54 Phe Phe Leu Phe Gly Leu Ala Leu Ile Val Gln Met Asp Ile Glu Gly 1 5 10 15 Thr Arg 55 18 PRT Lactuca sativa 55 Phe Phe Leu Phe Gly Leu Ser His Ile Val Leu Met Asp Leu Glu Gly 1 5 10 15 Thr Arg 56 18 PRT Adonis aestivalis 56 Phe Phe Leu Phe Gly Leu Glu Leu Ile Val Gln Leu Asp Ile Glu Ala 1 5 10 15 Thr Arg 57 18 PRT Arabidopsis sp. 57 Phe Phe Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Asp Ala 1 5 10 15 Thr Arg 58 411 PRT Synechococcus sp. 58 Met Phe Asp Ala Leu Val Ile Gly Ser Gly Pro Ala Gly Leu Ala Ile 1 5 10 15 Ala Ala Glu Leu Ala Gln Arg Gly Leu Lys Val Gln Gly Leu Ser Pro 20 25 30 Val Asp Pro Phe His Pro Trp Glu Asn Thr Tyr Gly Ile Trp Gly Pro 35 40 45 Glu Leu Asp Ser Leu Gly Leu Glu His Leu Phe Gly His Arg Trp Ser 50 55 60 Asn Cys Val Ser Tyr Phe Gly Glu Ala Pro Val Gln His Gln Tyr Asn 65 70 75 80 Tyr Gly Leu Phe Asp Arg Ala Gln Leu Gln Gln His Trp Leu Arg Gln 85 90 95 Cys Glu Gln Gly Gly Leu Gln Trp Gln Leu Gly Lys Ala Ala Ala Ile 100 105 110 Ala His Asp Ser His His Ser Cys Val Thr Thr Ala Ala Gly Gln Glu 115 120 125 Leu Gln Ala Arg Leu Val Val Asp Thr Thr Gly His Gln Ala Ala Phe 130 135 140 Ile Gln Arg Pro His Ser Asp Ala Ile Ala Tyr Gln Ala Ala Tyr Gly 145 150 155 160 Ile Ile Gly Gln Phe Ser Gln Pro Pro Ile Glu Pro His Gln Phe Val 165 170 175 Leu Met Asp Tyr Arg Ser Asp His Leu Ser Pro Glu Glu Arg Gln Leu 180 185 190 Pro Pro Thr Phe Leu Tyr Ala Met Asp Leu Gly Asn Asp Val Tyr Phe 195 200 205 Val Glu Glu Thr Ser Leu Ala Ala Cys Pro Ala Ile Pro Tyr Asp Arg 210 215 220 Leu Lys Gln Arg Leu Tyr Gln Arg Leu Ala Thr Arg Gly Val Thr Val 225 230 235 240 Gln Val Ile Gln His Glu Glu Tyr Cys Leu Phe Pro Met Asn Leu Pro 245 250 255 Leu Pro Asp Leu Thr Gln Ser Val Val Gly Phe Gly Gly Ala Ala Ser 260 265 270 Met Val His Pro Ala Ser Gly Tyr Met Val Gly Ala Leu Leu Arg Arg 275 280 285 Ala Pro Asp Leu Ala Asn Ala Ile Ala Ala Gly Leu Asn Ala Ser Ser 290 295 300 Ser Leu Thr Thr Ala Glu Leu Ala Thr Gln Ala Trp Arg Gly Leu Trp 305 310 315 320 Pro Thr Glu Lys Ile Arg Lys His Tyr Ile Tyr Gln Phe Gly Leu Glu 325 330 335 Lys Leu Met Arg Phe Ser Glu Ala Gln Leu Asn His His Phe Gln Thr 340 345 350 Phe Phe Gly Leu Pro Lys Glu Gln Trp Tyr Gly Phe Leu Thr Asn Thr 355 360 365 Leu Ser Leu Pro Glu Leu Ile Gln Ala Met Leu Arg Leu Phe Ala Gln 370 375 380 Ala Pro Asn Asp Val Arg Trp Gly Leu Met Glu Gln Gln Gly Arg Glu 385 390 395 400 Leu Gln Leu Phe Trp Gln Ala Ile Ala Ala Arg 405 410 59 420 PRT Arabidopsis thaliana 59 Val Val Asp Leu Ala Ile Val Gly Gly Gly Pro Ala Gly Leu Ala Val 1 5 10 15 Ala Gln Gln Val Ser Glu Ala Gly Leu Ser Val Cys Ser Ile Asp Pro 20 25 30 Ser Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Thr Thr Trp Ser Gly 50 55 60 Ala Val Val Tyr Val Asp Glu Gly Val Lys Lys Asp Leu Ser Arg Pro 65 70 75 80 Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Leu Gln Lys 85 90 95 Cys Ile Thr Asn Gly Val Lys Phe His Gln Ser Lys Val Thr Asn Val 100 105 110 Val His Glu Glu Ala Asn Ser Thr Val Val Cys Ser Asp Gly Val Lys 115 120 125 Ile Gln Ala Ser Val Val Leu Asp Ala Thr Gly Phe Ser Arg Cys Leu 130 135 140 Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala Tyr Gly 145 150 155 160 Ile Val Ala Glu Val Asp Gly His Pro Phe Asp Val Asp Lys Met Val 165 170 175 Phe Met Asp Trp Arg Asp Lys His Leu Asp Ser Tyr Pro Glu Leu Lys 180 185 190 Glu Arg Asn Ser Lys Ile Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser 195 200 205 Ser Asn Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly 210 215 220 Leu Arg Met Glu Asp Ile Gln Glu Arg Met Ala Ala Arg Leu Lys His 225 230 235 240 Leu Gly Ile Asn Val Lys Arg Ile Glu Glu Asp Glu Arg Cys Val Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Val Gly Ile 260 265 270 Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Leu Ala Ala Ala Pro Ile Val Ala Asn Ala Ile Val Arg Tyr 290 295 300 Leu Gly Ser Pro Ser Ser Asn Ser Leu Arg Gly Asp Gln Leu Ser Ala 305 310 315 320 Glu Val Trp Arg Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu 325 330 335 Phe Phe Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Asp Ala 340 345 350 Thr Arg Arg Phe Phe Asp Ala Phe Phe Asp Leu Gln Pro His Tyr Trp 355 360 365 His Gly Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Leu Val Phe 370 375 380 Gly Leu Ser Leu Phe Ser His Ala Ser Asn Thr Ser Arg Leu Glu Ile 385 390 395 400 Met Thr Lys Gly Thr Val Pro Leu Ala Lys Met Ile Asn Asn Leu Val 405 410 415 Gln Asp Arg Asp 420 60 418 PRT Lycopersicon esculentum 60 Val Val Asp Leu Ala Val Val Gly Gly Gly Pro Ala Gly Leu Ala Val 1 5 10 15 Ala Gln Gln Val Ser Glu Ala Gly Leu Ser Val Cys Ser Ile Asp Pro 20 25 30 Asn Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Ala Thr Trp Ser Gly 50 55 60 Ala Ala Val Tyr Ile Asp Asp Asn Thr Ala Lys Asp Leu His Arg Pro 65 70 75 80 Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Met Gln Lys 85 90 95 Cys Ile Met Asn Gly Val Lys Phe His Gln Ala Lys Val Ile Lys Val 100 105 110 Ile His Glu Glu Ser Lys Ser Met Leu Ile Cys Asn Asp Gly Ile Thr 115 120 125 Ile Gln Ala Thr Val Val Leu Asp Ala Thr Gly Phe Ser Arg Ser Leu 130 135 140 Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala Tyr Gly 145 150 155 160 Ile Leu Ala Glu Val Glu Glu His Pro Phe Asp Val Asn Lys Met Val 165 170 175 Phe Met Asp Trp Arg Asp Ser His Leu Lys Asn Asn Thr Asp Leu Lys 180 185 190 Glu Arg Asn Ser Arg Ile Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser 195 200 205 Ser Asn Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly 210 215 220 Leu Arg Ile Asp Asp Ile Gln Glu Arg Met Val Ala Arg Leu Asn His 225 230 235 240 Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu His Cys Leu Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Val Gly Ile 260 265 270 Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Leu Ala Ala Ala Pro Val Val Ala Asn Ala Ile Ile Gln Tyr 290 295 300 Leu Gly Ser Glu Arg Ser His Ser Gly Asn Glu Leu Ser Thr Ala Val 305 310 315 320 Trp Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu Phe Phe 325 330 335 Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Pro Ala Thr Arg 340 345 350 Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg Tyr Trp His Gly 355 360 365 Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Ile Val Phe Gly Leu 370 375 380 Ser Leu Phe Ser His Ala Ser Asn Thr Ser Arg Phe Glu Ile Met Thr 385 390 395 400 Lys Gly Thr Val Pro Leu Val Asn Met Ile Asn Asn Leu Leu Gln Asp 405 410 415 Lys Glu 61 418 PRT Capsicum annuum 61 Val Val Asp Leu Ala Val Val Gly Gly Gly Pro Ala Gly Leu Ala Val 1 5 10 15 Ala Gln Gln Val Ser Glu Ala Gly Leu Ser Val Cys Ser Ile Asp Pro 20 25 30 Asn Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Ala Thr Trp Ser Gly 50 55 60 Ala Ala Val Tyr Ile Asp Asp Lys Thr Thr Lys Asp Leu Asn Arg Pro 65 70 75 80 Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Met Gln Lys 85 90 95 Cys Ile Leu Asn Gly Val Lys Phe His Gln Ala Lys Val Ile Lys Val 100 105 110 Ile His Glu Glu Ser Lys Ser Met Leu Ile Cys Asn Asp Gly Ile Thr 115 120 125 Ile Gln Ala Thr Val Val Leu Asp Ala Thr Gly Phe Ser Arg Ser Leu 130 135 140 Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala Tyr Gly 145 150 155 160 Ile Leu Ala Glu Val Glu Glu His Pro Phe Asp Val Asn Lys Met Val 165 170 175 Phe Met Asp Trp Arg Asp Ser His Leu Lys Asn Asn Val Glu Leu Lys 180 185 190 Glu Arg Asn Ser Arg Ile Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser 195 200 205 Ser Asn Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly 210 215 220 Leu Gly Met Asp Asp Ile Gln Glu Arg Met Val Ala Arg Leu Ser His 225 230 235 240 Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu His Cys Val Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Val Gly Ile 260 265 270 Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Leu Ala Ala Ala Pro Val Val Ala Asn Ala Ile Ile Gln Tyr 290 295 300 Leu Ser Ser Glu Arg Ser His Ser Gly Asp Glu Leu Ser Ala Ala Val 305 310 315 320 Trp Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu Phe Phe 325 330 335 Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Pro Ala Thr Arg 340 345 350 Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg Tyr Trp His Gly 355 360 365 Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Ile Val Phe Gly Leu 370 375 380 Ser Leu Phe Ser His Ala Ser Asn Thr Ser Arg Leu Glu Ile Met Thr 385 390 395 400 Lys Gly Thr Leu Pro Leu Val His Met Ile Asn Asn Leu Leu Gln Asp 405 410 415 Lys Glu 62 418 PRT Nicotiana tabacum 62 Val Val Asp Leu Ala Val Val Gly Gly Gly Pro Ala Gly Leu Ala Val 1 5 10 15 Ala Gln Gln Val Ser Glu Ala Gly Leu Ser Val Val Ser Ile Asp Pro 20 25 30 Ser Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Ala Thr Trp Ser Gly 50 55 60 Thr Val Val Tyr Ile Asp Asp Asn Thr Thr Lys Asp Leu Asp Arg Pro 65 70 75 80 Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Met Gln Lys 85 90 95 Cys Ile Leu Asn Gly Val Lys Phe His His Ala Lys Val Ile Lys Val 100 105 110 Ile His Glu Glu Ala Lys Ser Met Leu Ile Cys Asn Asp Gly Val Thr 115 120 125 Ile Gln Ala Thr Val Val Leu Asp Ala Thr Gly Phe Ser Arg Cys Leu 130 135 140 Val Gln Tyr Asp Lys Pro Tyr Lys Pro Gly Tyr Gln Val Ala Tyr Gly 145 150 155 160 Ile Leu Ala Glu Val Glu Glu His Pro Phe Asp Thr Ser Lys Met Val 165 170 175 Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Asn Met Glu Leu Lys 180 185 190 Glu Arg Asn Arg Lys Val Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser 195 200 205 Ser Asn Lys Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly 210 215 220 Leu Arg Met Asp Asp Ile Gln Glu Arg Met Val Ala Arg Leu Asn His 225 230 235 240 Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu His Cys Val Ile 245 250 255 Pro Met Gly Gly Ser Leu Pro Val Ile Pro Gln Arg Val Val Gly Thr 260 265 270 Gly Gly Thr Ala Gly Leu Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Leu Ala Ala Ala Pro Val Val Ala Asn Ala Ile Ile His Tyr 290 295 300 Leu Gly Ser Glu Lys Asp Leu Leu Gly Asn Glu Leu Ser Ala Ala Val 305 310 315 320 Trp Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu Phe Phe 325 330 335 Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Pro Ala Thr Arg 340 345 350 Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg Tyr Trp His Gly 355 360 365 Phe Leu Ser Ser Arg Leu Tyr Leu Pro Glu Leu Ile Phe Phe Gly Leu 370 375 380 Ser Leu Phe Ser Arg Ala Ser Asn Thr Ser Arg Ile Glu Ile Met Thr 385 390 395 400 Lys Gly Thr Leu Pro Leu Val Asn Met Ile Asn Asn Leu Leu Gln Asp 405 410 415 Thr Glu 63 418 PRT Adonis palaestina 63 Val Val Asp Leu Ala Val Val Gly Gly Gly Pro Ala Gly Leu Ala Ile 1 5 10 15 Ala Gln Gln Val Ser Glu Ala Gly Leu Leu Val Cys Ser Ile Asp Pro 20 25 30 Ser Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Thr Thr Trp Ser Gly 50 55 60 Ala Val Val Tyr Thr Asp Asp Asn Ser Lys Lys Tyr Leu Asp Arg Pro 65 70 75 80 Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Leu Gln Lys 85 90 95 Cys Val Thr Asn Gly Val Lys Phe His Gln Ala Lys Val Ile Lys Val 100 105 110 Ile His Glu Glu Ser Lys Ser Leu Leu Ile Cys Asn Asp Gly Ile Thr 115 120 125 Ile Asn Ala Thr Val Val Leu Asp Ala Thr Gly Phe Ser Arg Cys Leu 130 135 140 Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala Tyr Gly 145 150 155 160 Ile Met Ala Glu Val Glu Glu His Pro Phe Asp Leu Asp Lys Met Leu 165 170 175 Phe Met Asp Trp Arg Asp Ser His Leu Asn Glu Lys Leu Glu Leu Lys 180 185 190 Asp Lys Asn Arg Lys Ile Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser 195 200 205 Ser Thr Lys Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly 210 215 220 Leu Arg Phe Glu Asp Ile Gln Glu Arg Met Val Ala Arg Leu Lys His 225 230 235 240 Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu Arg Cys Val Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Val Gly Ile 260 265 270 Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Leu Ala Ala Ala Pro Val Val Ala Lys Ser Ile Val Gln Tyr 290 295 300 Leu Gly Ser Asp Arg Ser Leu Ser Gly Asn Glu Leu Ser Ala Glu Val 305 310 315 320 Trp Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu Phe Phe 325 330 335 Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Gln Gly Thr Arg 340 345 350 Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro His Tyr Trp His Gly 355 360 365 Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Leu Phe Phe Gly Leu 370 375 380 Ser Leu Phe Ser His Ala Ser Asn Ala Ser Arg Ile Glu Ile Met Ala 385 390 395 400 Lys Gly Thr Val Pro Leu Val Asn Met Met Asn Asn Leu Ile Gln Asp 405 410 415 Thr Asp 64 422 PRT Tagetes erecta 64 Val Val Asp Leu Val Val Val Gly Gly Gly Pro Ser Gly Leu Ala Val 1 5 10 15 Ala Gln Gln Val Ser Glu Ala Gly Leu Thr Val Cys Ser Ile Asp Pro 20 25 30 Ser Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Thr Thr Trp Ser Ser 50 55 60 Ala Val Val Tyr Ile Asp Glu Lys Ser Thr Lys Ser Leu Asn Arg Pro 65 70 75 80 Tyr Ala Arg Val Asn Arg Lys Gln Leu Lys Thr Lys Met Leu Gln Lys 85 90 95 Cys Ile Ala Asn Gly Val Lys Phe His Gln Ala Lys Val Ile Lys Val 100 105 110 Ile His Glu Glu Leu Lys Ser Leu Leu Ile Cys Asn Asp Gly Val Thr 115 120 125 Ile Gln Ala Thr Leu Val Leu Asp Ala Thr Gly Phe Ser Arg Ser Leu 130 135 140 Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala Tyr Gly 145 150 155 160 Ile Leu Ala Glu Val Glu Glu His Pro Phe Asp Val Asp Lys Met Leu 165 170 175 Phe Met Asp Trp Arg Asp Ser His Leu Asp Gln Asn Leu Glu Ile Lys 180 185 190 Ala Arg Asn Ser Arg Ile Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser 195 200 205 Ser Thr Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly 210 215 220 Leu Lys Met Glu Asp Ile Gln Glu Arg Met Ala Tyr Arg Leu Lys His 225 230 235 240 Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu Arg Cys Val Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Leu Gly Ile 260 265 270 Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Leu Ala Ala Ala Pro Ile Val Ala Lys Ser Ile Ile Arg Tyr 290 295 300 Leu Asn Asn Glu Lys Ser Met Val Ala Asp Val Thr Gly Asp Asp Leu 305 310 315 320 Ala Ala Gly Ile Trp Arg Glu Leu Trp Pro Ile Glu Arg Arg Arg Gln 325 330 335 Arg Glu Phe Phe Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu 340 345 350 Glu Gly Thr Arg Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg 355 360 365 Tyr Trp His Gly Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Val 370 375 380 Thr Phe Gly Leu Ser Leu Phe Gly His Ala Ser Asn Thr Cys Arg Val 385 390 395 400 Glu Ile Met Ala Lys Gly Thr Leu Pro Leu Ala Thr Met Ile Gly Asn 405 410 415 Leu Val Arg Asp Arg Glu 420 65 417 PRT Narcissus pseudonarcissus 65 Thr Leu Asp Leu Ala Val Val Gly Gly Gly Pro Leu Ala Arg Ser Cys 1 5 10 15 Ser Thr Ser Leu Gly Gly Gly Leu Ser Val Val Ser Ile Asp Pro Asn 20 25 30 Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu Phe 35 40 45 Glu Asp Met Asp Leu Leu Asp Cys Leu Asp Ala Thr Trp Ser Gly Ala 50 55 60 Ile Val Tyr Val Asp Asp Arg Ser Thr Lys Asn Leu Ser Arg Pro Tyr 65 70 75 80 Ala Arg Val Asn Arg Lys Asn Leu Lys Ser Lys Met Met Lys Lys Cys 85 90 95 Val Ser Asn Gly Val Arg Phe His Gln Ala Thr Val Val Lys Ala Met 100 105 110 His Glu Glu Glu Lys Ser Tyr Leu Ile Cys Ser Asp Gly Val Thr Ile 115 120 125 Asp Ala Arg Val Val Leu Asp Ala Thr Gly Phe Ser Arg Cys Leu Val 130 135 140 Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala Tyr Gly Ile 145 150 155 160 Leu Ala Glu Val Glu Glu His Pro Phe Asp Val Asp Lys Met Val Phe 165 170 175 Met Asp Trp Arg Asp Ser His Leu Asn Gly Lys Ala Glu Leu Asn Glu 180 185 190 Arg Asn Ala Lys Ile Pro Thr Phe Leu Tyr Ala Met Pro Phe Ser Ser 195 200 205 Asn Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg Pro Gly Leu 210 215 220 Lys Met Glu Asp Ile Gln Glu Arg Met Val Ala Arg Leu Asn His Leu 225 230 235 240 Gly Ile Arg Ile Lys Ser Ile Glu Glu Asp Glu Arg Cys Val Ile Pro 245 250 255 Met Gly Gly Pro Leu Pro Val Ile Pro Gln Arg Val Val Gly Ile Gly 260 265 270 Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met Val Ala Arg 275 280 285 Thr Leu Ala Ala Ala Pro Ile Val Ala Asn Ser Ile Val Gln Tyr Leu 290 295 300 Val Ser Asp Ser Gly Leu Ser Gly Asn Asp Leu Ser Ala Asp Val Trp 305 310 315 320 Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu Phe Phe Cys 325 330 335 Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Glu Gly Thr Arg Arg 340 345 350 Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg Tyr Trp His Gly Phe 355 360 365 Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Val Pro Phe Gly Leu Ser 370 375 380 Leu Phe Ser His Ala Ser Asn Thr Cys Lys Leu Glu Ile Met Ala Lys 385 390 395 400 Gly Thr Leu Pro Leu Val Asn Met Ile Asn Asn Leu Val Gln Asp Arg 405 410 415 Asp 66 418 PRT Solanum tuberosum 66 Gln Phe Asp Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Arg Leu 1 5 10 15 Ala Glu His Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp Pro 20 25 30 Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Asn Leu Gly Leu Glu Asp Cys Leu Asp His Lys Trp Pro Met 50 55 60 Thr Cys Val His Ile Asn Asp His Lys Thr Lys Tyr Leu Gly Arg Pro 65 70 75 80 Tyr Gly Arg Val Ser Arg Lys Lys Leu Lys Leu Arg Leu Leu Asn Ser 85 90 95 Cys Val Glu Asn Arg Val Lys Phe Tyr Lys Ala Lys Val Trp Lys Val 100 105 110 Glu His Glu Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Lys Lys 115 120 125 Ile Arg Gly Ser Leu Val Val Asp Ala Ser Gly Phe Ala Ser Asp Phe 130 135 140 Ile Glu Tyr Asp Lys Pro Arg Asn His Gly Tyr Gln Ile Ala His Gly 145 150 155 160 Val Leu Val Glu Val Asp Asn His Pro Phe Asp Leu Asp Lys Met Val 165 170 175 Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu Arg 180 185 190 Val Asn Asn Ala Lys Glu Pro Thr Phe Leu Tyr Ala Met Pro Phe Asp 195 200 205 Arg Asn Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro Val 210 215 220 Leu Ser Tyr Met Glu Val Lys Arg Arg Met Val Ala Arg Leu Arg His 225 230 235 240 Leu Gly Ile Lys Val Arg Ser Val Ile Glu Glu Glu Lys Cys Val Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Arg Ile Pro Gln Asn Val Met Ala Ile 260 265 270 Gly Gly Asn Ser Gly Ile Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Ser Met Ala Leu Ala Pro Val Leu Ala Glu Ala Ile Val Lys Gly 290 295 300 Leu Gly Ser Thr Arg Met Ile Arg Gly Ser Gln Leu Tyr His Arg Val 305 310 315 320 Trp Asn Gly Leu Trp Pro Leu Asp Arg Arg Cys Ile Gly Glu Cys Tyr 325 330 335 Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr Arg 340 345 350 Arg Leu Phe Asp Ala Phe Phe Asp Leu Asp Pro Lys Tyr Trp Gln Gly 355 360 365 Phe Leu Ser Ser Arg Leu Ser Val Lys Glu Leu Ala Ile Leu Ser Leu 370 375 380 Cys Leu Phe Gly His Gly Ser Asn Leu Thr Arg Leu Asp Ile Val Thr 385 390 395 400 Lys Cys Pro Val Pro Leu Val Arg Leu Ile Gly Asn Leu Ala Ile Glu 405 410 415 Ser Leu 67 418 PRT Lycopersicon esculentum 67 Gln Phe Asp Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Arg Leu 1 5 10 15 Ala Glu Gln Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp Pro 20 25 30 Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Asn Leu Gly Leu Glu Asp Cys Leu Asp His Lys Trp Pro Met 50 55 60 Thr Cys Val His Ile Asn Asp Asn Lys Thr Lys Tyr Leu Gly Arg Pro 65 70 75 80 Tyr Gly Arg Val Ser Arg Lys Lys Leu Lys Leu Lys Leu Leu Asn Ser 85 90 95 Cys Val Glu Asn Arg Val Lys Phe Tyr Lys Ala Lys Val Trp Lys Val 100 105 110 Glu His Glu Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Lys Lys 115 120 125 Ile Arg Gly Ser Leu Val Val Asp Ala Ser Asp Phe Ala Ser Asp Phe 130 135 140 Ile Glu Tyr Asp Arg Pro Arg Asn His Gly Tyr Gln Ile Ala His Gly 145 150 155 160 Val Leu Val Glu Val Asp Asn His Pro Phe Asp Leu Asp Lys Met Val 165 170 175 Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu Arg 180 185 190 Val Asn Asn Ala Lys Glu Pro Thr Phe Leu Tyr Ala Met Pro Phe Asp 195 200 205 Arg Asp Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro Val 210 215 220 Leu Ser Tyr Met Glu Val Lys Arg Arg Met Val Ala Arg Leu Arg His 225 230 235 240 Leu Gly Ile Lys Val Lys Ser Val Ile Glu Glu Glu Lys Cys Val Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Arg Ile Pro Gln Asn Val Met Ala Ile 260 265 270 Gly Gly Asn Ser Gly Ile Val His Pro Ser Thr Gly Tyr Met Val Ala 275 280 285 Arg Ser Met Ala Leu Ala Pro Val Leu Ala Glu Ala Ile Val Glu Gly 290 295 300 Leu Gly Ser Thr Arg Met Ile Arg Gly Ser Gln Leu Tyr His Arg Val 305 310 315 320 Trp Asn Gly Leu Trp Pro Leu Asp Arg Arg Cys Val Arg Glu Cys Tyr 325 330 335 Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr Arg 340 345 350 Arg Leu Phe Asp Ala Phe Phe Asp Leu Asp Pro Lys Tyr Trp Gln Gly 355 360 365 Phe Leu Ser Ser Arg Leu Ser Val Lys Glu Leu Gly Leu Leu Ser Leu 370 375 380 Cys Leu Phe Gly His Gly Ser Asn Met Thr Arg Leu Asp Ile Val Thr 385 390 395 400 Lys Cys Pro Leu Pro Leu Val Arg Leu Met Gly Asn Leu Ala Ile Glu 405 410 415 Ser Leu 68 419 PRT Citrus sinensis 68 Arg Tyr Asp Val Ile Ile Ile Gly Thr Gly Pro Ala Gly Leu Arg Leu 1 5 10 15 Ala Glu Gln Val Ser Ser Arg His Ser Val Lys Val Cys Cys Val Asp 20 25 30 Pro Ser Pro Leu Ser Thr Trp Pro Asn Asn Tyr Gly Val Trp Val Asp 35 40 45 Glu Phe Glu Asp Ile Gly Leu Val Asp Cys Leu Asp Lys Thr Trp Pro 50 55 60 Met Thr Cys Val Phe Ile Asn Asp His Lys Thr Lys Tyr Leu Asp Arg 65 70 75 80 Pro Tyr Gly Arg Val Ser Arg Asn Ile Leu Lys Thr Lys Leu Leu Glu 85 90 95 Asn Cys Val Ser Asn Gly Val Lys Phe His Lys Ala Lys Val Trp His 100 105 110 Val Asn His Gln Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Asn 115 120 125 Glu Ile Lys Ala Ser Leu Ile Val Asp Ala Ser Gly Phe Ala Ser Ser 130 135 140 Phe Val Glu Tyr Asp Lys Pro Arg Asn His Gly Tyr Gln Ile Ala His 145 150 155 160 Gly Ile Leu Ala Glu Val Glu Ser His Pro Phe Asp Leu Asp Lys Met 165 170 175 Val Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu 180 185 190 Arg Ala Ser Asn Leu Lys Leu Pro Thr Phe Leu Tyr Ala Met Pro Phe 195 200 205 Asp Ser Asn Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro 210 215 220 Val Leu Ser Tyr Lys Glu Val Lys Ser Arg Met Ala Ala Arg Leu Arg 225 230 235 240 His Met Gly Ile Arg Val Lys Arg Val Ile Glu Asp Glu Lys Cys Leu 245 250 255 Ile Pro Met Gly Gly Pro Leu Pro Val Ile Pro Gln Ser Val Met Ala 260 265 270 Ile Gly Gly Thr Ser Gly Leu Ile His Pro Ala Thr Gly Tyr Met Val 275 280 285 Ala Arg Thr Met Ala Leu Ala Pro Ala Leu Ala Asp Ala Ile Ala Glu 290 295 300 Cys Leu Gly Ser Thr Arg Met Ile Arg Gly Arg Pro Leu His Gln Lys 305 310 315 320 Val Trp Asn Gly Leu Trp Pro Ile Asp Arg Arg Cys Asn Arg Glu Phe 325 330 335 Tyr Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr 340 345 350 Arg Arg Phe Phe Asp Ala Phe Phe Asp Leu Asn Pro Tyr Tyr Trp His 355 360 365 Gly Phe Leu Ser Ser Arg Leu Ser Leu Ala Glu Leu Ala Gly Leu Ser 370 375 380 Leu Ser Leu Phe Gly His Ala Ser Asn Ser Ser Arg Leu Asp Ile Val 385 390 395 400 Thr Lys Cys Pro Val Pro Leu Val Lys Met Met Gly Asn Leu Ala Leu 405 410 415 Glu Thr Ile 69 418 PRT Capsicum annuum 69 Glu Phe Asp Val Ile Ile Ile Gly Thr Gly Pro Ala Gly Leu Arg Leu 1 5 10 15 Ala Glu Gln Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp Pro 20 25 30 Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu 35 40 45 Phe Glu Lys Leu Gly Leu Glu Asp Cys Leu Asp His Lys Trp Pro Val 50 55 60 Ser Cys Val His Ile Ser Asp His Lys Thr Lys Tyr Leu Asp Arg Pro 65 70 75 80 Tyr Gly Arg Val Ser Arg Asn Ile Leu Lys Thr Lys Leu Leu Glu Asn 85 90 95 Cys Val Ser Asn Gly Val Lys Phe His Lys Ala Lys Val Trp His Val 100 105 110 Asn His Gln Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Asn Glu 115 120 125 Ile Lys Ala Ser Leu Ile Val Asp Ala Ser Gly Phe Ala Ser Ser Phe 130 135 140 Val Glu Tyr Asp Lys Pro Arg Asn His Gly Tyr Gln Ile Ala His Gly 145 150 155 160 Ile Leu Ala Glu Val Glu Ser His Pro Phe Asp Leu Asp Lys Met Val 165 170 175 Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu Arg 180 185 190 Ala Ser Asn Leu Lys Leu Pro Thr Phe Leu Tyr Ala Met Pro Phe Asp 195 200 205 Ser Asn Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro Val 210 215 220 Leu Ser Tyr Lys Glu Val Lys Ser Arg Met Ala Ala Arg Leu Arg His 225 230 235 240 Met Gly Ile Arg Val Lys Arg Val Ile Glu Asp Glu Lys Cys Leu Ile 245 250 255 Pro Met Gly Gly Pro Leu Pro Val Ile Pro Gln Ser Val Met Ala Ile 260 265 270 Gly Gly Thr Ser Gly Leu Ile His Pro Ala Thr Gly Tyr Met Val Ala 275 280 285 Arg Thr Met Ala Leu Ala Pro Ala Leu Ala Asp Ala Ile Ala Glu Cys 290 295 300 Leu Gly Ser Thr Arg Met Ile Arg Gly Arg Pro Leu His Gln Lys Val 305 310 315 320 Trp Asn Gly Leu Trp Pro Ile Asp Arg Arg Cys Asn Arg Glu Phe Tyr 325 330 335 Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr Arg 340 345 350 Arg Phe Phe Asp Ala Phe Phe Asp Leu Asn Pro Tyr Tyr Trp His Gly 355 360 365 Phe Leu Ser Ser Arg Leu Ser Leu Ala Glu Leu Ala Gly Leu Ser Leu 370 375 380 Ser Leu Phe Gly His Ala Ser Asn Ser Ser Arg Leu Asp Ile Val Thr 385 390 395 400 Lys Cys Pro Val Pro Leu Val Lys Met Met Gly Asn Leu Ala Leu Glu 405 410 415 Thr Ile 70 422 PRT Lactuca sativa 70 Ile Leu Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ala Leu 1 5 10 15 Ala Ala Glu Ser Ala Lys Leu Gly Leu Asn Val Gly Leu Ile Gly Pro 20 25 30 Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Gln Asp Glu Phe Ile 35 40 45 Gly Leu Gly Leu Glu Gly Cys Ile Glu His Ser Trp Lys Asp Thr Leu 50 55 60 Val Tyr Leu Asp Asp Ala Asp Pro Ile Arg Ile Gly Arg Ala Tyr Gly 65 70 75 80 Arg Val His Arg Asp Leu Leu His Glu Glu Leu Leu Arg Arg Cys Val 85 90 95 Glu Ser Gly Val Ser Tyr Leu Ser Ser Lys Val Glu Arg Ile Thr Glu 100 105 110 Ala Pro Asn Gly Tyr Ser Leu Ile Glu Cys Glu Gly Asn Ile Thr Ile 115 120 125 Pro Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Phe 130 135 140 Leu Glu Tyr Glu Leu Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr 145 150 155 160 Gly Ile Glu Val Glu Val Glu Asn Asn Pro Tyr Asp Pro Asp Leu Met 165 170 175 Val Phe Met Asp Tyr Arg Asp Phe Ser Lys His Lys Pro Glu Ser Leu 180 185 190 Glu Ala Lys Tyr Pro Thr Phe Leu Tyr Val Met Ala Met Ser Pro Thr 195 200 205 Lys Ile Phe Phe Glu Glu Thr Cys Leu Ala Ser Arg Glu Ala Met Pro 210 215 220 Phe Asn Leu Leu Lys Ser Lys Leu Met Ser Arg Leu Lys Ala Met Gly 225 230 235 240 Ile Arg Ile Thr Arg Thr Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val 245 250 255 Gly Gly Ser Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala 260 265 270 Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser 275 280 285 Leu Ser Glu Ala Pro Asn Tyr Ala Ala Val Ile Ala Lys Ile Leu Arg 290 295 300 Gln Asp Gln Ser Lys Glu Met Ile Ser Leu Gly Lys Tyr Thr Asn Ile 305 310 315 320 Ser Lys Gln Ala Trp Glu Thr Leu Trp Pro Leu Glu Arg Lys Arg Gln 325 330 335 Arg Ala Phe Phe Leu Phe Gly Leu Ser His Ile Val Leu Met Asp Leu 340 345 350 Glu Gly Thr Arg Thr Phe Phe Arg Thr Phe Phe Arg Leu Pro Lys Trp 355 360 365 Met Trp Trp Gly Phe Leu Gly Ser Ser Leu Ser Ser Thr Asp Leu Ile 370 375 380 Ile Phe Ala Leu Tyr Met Phe Val Ile Ala Pro His Ser Leu Arg Met 385 390 395 400 Glu Leu Val Arg His Leu Leu Ser Asp Pro Thr Gly Ala Thr Met Val 405 410 415 Lys Ala Tyr Leu Thr Ile 420 71 416 PRT Arabidopsis thaliana 71 Ala Leu Asp His Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ala Leu 1 5 10 15 Ala Ala Glu Ser Ala Lys Leu Gly Leu Lys Val Gly Leu Ile Gly Pro 20 25 30 Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Asn 35 40 45 Asp Leu Gly Leu Gln Lys Cys Ile Glu His Val Trp Arg Glu Thr Ile 50 55 60 Val Tyr Leu Asp Asp Asp Lys Pro Ile Thr Ile Gly Arg Ala Tyr Gly 65 70 75 80 Arg Val Ser Arg Arg Leu His Glu Glu Leu Leu Arg Arg Cys Val Glu 85 90 95 Ser Gly Val Ser Tyr Leu Ser Ser Lys Val Asp Ser Ile Thr Glu Ala 100 105 110 Ser Asp Gly Leu Arg Leu Val Ala Cys Asp Asp Asn Asn Val Ile Pro 115 120 125 Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Leu Leu 130 135 140 Gln Tyr Glu Val Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr Gly 145 150 155 160 Val Glu Val Glu Val Glu Asn Ser Pro Tyr Asp Pro Asp Gln Met Val 165 170 175 Phe Met Asp Tyr Arg Asp Tyr Thr Asn Glu Lys Val Arg Ser Leu Glu 180 185 190 Ala Glu Tyr Pro Thr Phe Leu Tyr Ala Met Pro Met Thr Lys Ser Arg 195 200 205 Leu Phe Phe Glu Glu Thr Cys Leu Ala Ser Lys Asp Val Met Pro Phe 210 215 220 Asp Leu Leu Lys Thr Lys Leu Met Leu Arg Leu Asp Thr Leu Gly Ile 225 230 235 240 Arg Ile Leu Lys Thr Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val Gly 245 250 255 Gly Ser Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala Ala 260 265 270 Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser Leu 275 280 285 Ser Glu Ala Pro Lys Tyr Ala Ser Val Ile Ala Glu Ile Leu Arg Glu 290 295 300 Glu Thr Thr Lys Gln Ile Asn Ser Asn Ile Ser Arg Gln Ala Trp Asp 305 310 315 320 Thr Leu Trp Pro Pro Glu Arg Lys Arg Gln Arg Ala Phe Phe Leu Phe 325 330 335 Gly Leu Ala Leu Ile Val Gln Phe Asp Thr Glu Gly Ile Arg Ser Phe 340 345 350 Phe Arg Thr Phe Phe Arg Leu Pro Lys Trp Met Trp Gln Gly Phe Leu 355 360 365 Gly Ser Thr Leu Thr Ser Gly Asp Leu Val Leu Phe Ala Leu Tyr Met 370 375 380 Phe Val Ile Ser Pro Asn Asn Leu Arg Lys Gly Leu Ile Asn His Leu 385 390 395 400 Ile Ser Asp Pro Thr Gly Ala Thr Met Ile Lys Thr Tyr Leu Lys Val 405 410 415 72 423 PRT Adonis palaestina 72 Val Met Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ser Leu 1 5 10 15 Ala Ala Glu Ala Ala Lys Leu Gly Leu Lys Val Gly Leu Ile Gly Pro 20 25 30 Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Lys 35 40 45 Asp Leu Gly Leu Glu Arg Cys Ile Glu His Ala Trp Lys Asp Thr Ile 50 55 60 Val Tyr Leu Asp Asn Asp Ala Pro Val Leu Ile Gly Arg Ala Tyr Gly 65 70 75 80 Arg Val Ser Arg His Leu Leu His Glu Glu Leu Leu Lys Arg Cys Val 85 90 95 Glu Ser Gly Val Ser Tyr Leu Asp Ser Lys Val Glu Arg Ile Thr Glu 100 105 110 Ala Gly Asp Gly His Ser Leu Val Val Cys Glu Asn Glu Ile Phe Ile 115 120 125 Pro Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Leu 130 135 140 Leu Glu Tyr Glu Val Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr 145 150 155 160 Gly Val Glu Val Glu Val Glu Asn Asn Pro Tyr Asp Pro Asn Leu Met 165 170 175 Val Phe Met Asp Tyr Arg Asp Tyr Met Gln Gln Lys Leu Gln Cys Ser 180 185 190 Glu Glu Glu Tyr Pro Thr Phe Leu Tyr Val Met Pro Met Ser Pro Thr 195 200 205 Arg Leu Phe Phe Glu Glu Thr Cys Leu Ala Ser Lys Asp Ala Met Pro 210 215 220 Phe Asp Leu Leu Lys Arg Lys Leu Met Ser Arg Leu Lys Thr Leu Gly 225 230 235 240 Ile Gln Val Thr Lys Val Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val 245 250 255 Gly Gly Ser Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala 260 265 270 Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser 275 280 285 Leu Ser Glu Ala Pro Lys Tyr Ala Ser Val Ile Ala Lys Ile Leu Lys 290 295 300 Gln Asp Asn Ser Ala Tyr Val Val Ser Gly Gln Ser Ser Ala Val Asn 305 310 315 320 Ile Ser Met Gln Ala Trp Ser Ser Leu Trp Pro Lys Glu Arg Lys Arg 325 330 335 Gln Arg Ala Phe Phe Leu Phe Gly Leu Glu Leu Ile Val Gln Leu Asp 340 345 350 Ile Glu Ala Thr Arg Thr Phe Phe Arg Thr Phe Phe Arg Leu Pro Thr 355 360 365 Trp Met Trp Trp Gly Phe Leu Gly Ser Ser Leu Ser Ser Phe Asp Leu 370 375 380 Val Leu Phe Ser Met Tyr Met Phe Val Leu Ala Pro Asn Ser Met Arg 385 390 395 400 Met Ser Leu Val Arg His Leu Leu Ser Asp Pro Ser Gly Ala Val Met 405 410 415 Val Arg Ala Tyr Leu Glu Arg 420 73 423 PRT Adonis palaestina 73 Val Met Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ser Leu 1 5 10 15 Ala Ala Glu Ala Ala Lys Leu Gly Leu Lys Val Gly Leu Ile Gly Pro 20 25 30 Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Lys 35 40 45 Asp Leu Gly Leu Glu Arg Cys Ile Glu His Ala Trp Lys Asp Thr Ile 50 55 60 Val Tyr Leu Asp Asn Asp Ala Pro Val Leu Ile Gly Arg Ala Tyr Gly 65 70 75 80 Arg Val Ser Arg His Leu Leu His Glu Glu Leu Leu Lys Arg Cys Val 85 90 95 Glu Ser Gly Val Ser Tyr Leu Asn Ser Lys Val Glu Arg Ile Thr Glu 100 105 110 Ala Gly Asp Gly His Ser Leu Val Val Cys Glu Asn Asp Ile Phe Ile 115 120 125 Pro Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Leu 130 135 140 Leu Glu Tyr Glu Val Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr 145 150 155 160 Gly Val Glu Val Glu Val Glu Asn Asn Pro Tyr Asp Pro Asn Leu Met 165 170 175 Val Phe Met Asp Tyr Arg Asp Tyr Met Gln Gln Lys Leu Gln Cys Ser 180 185 190 Glu Glu Glu Tyr Pro Thr Phe Leu Tyr Val Met Pro Met Ser Pro Thr 195 200 205 Arg Leu Phe Phe Glu Glu Thr Cys Leu Ala Ser Lys Asp Ala Met Pro 210 215 220 Phe Asp Leu Leu Lys Arg Lys Leu Met Ser Arg Leu Lys Thr Leu Gly 225 230 235 240 Ile Gln Val Thr Lys Ile Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val 245 250 255 Gly Gly Ser Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala 260 265 270 Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser 275 280 285 Leu Ser Glu Ala Pro Lys Tyr Ala Ser Val Ile Ala Lys Ile Leu Lys 290 295 300 Gln Asp Asn Ser Ala Tyr Val Val Ser Gly Gln Ser Ser Ala Val Asn 305 310 315 320 Ile Ser Met Gln Ala Trp Ser Ser Leu Trp Pro Lys Glu Arg Lys Arg 325 330 335 Gln Arg Ala Phe Phe Leu Phe Gly Leu Glu Leu Ile Val Gln Leu Asp 340 345 350 Ile Glu Ala Thr Arg Thr Phe Phe Arg Thr Phe Phe Arg Leu Pro Thr 355 360 365 Trp Met Trp Trp Gly Phe Leu Gly Ser Ser Leu Ser Ser Phe Asp Leu 370 375 380 Val Leu Phe Ser Met Tyr Met Phe Val Leu Ala Pro Asn Ser Met Arg 385 390 395 400 Met Ser Leu Val Arg His Leu Leu Ser Asp Pro Ser Gly Ala Val Met 405 410 415 Val Lys Ala Tyr Leu Glu Arg 420 74 423 PRT Tagetes erecta 74 Ile Leu Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ala Leu 1 5 10 15 Ala Gly Glu Ser Ala Lys Leu Gly Leu Asn Val Ala Leu Ile Gly Pro 20 25 30 Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Ile 35 40 45 Gly Leu Gly Leu Glu Gly Cys Ile Glu His Val Trp Arg Asp Thr Val 50 55 60 Val Tyr Leu Asp Asp Asn Asp Pro Ile Leu Ile Gly Arg Ala Tyr Gly 65 70 75 80 Arg Val Ser Arg Asp Leu Leu His Glu Glu Leu Leu Thr Arg Cys Met 85 90 95 Glu Ser Gly Val Ser Tyr Leu Ser Ser Lys Val Glu Arg Ile Thr Glu 100 105 110 Ala Pro Asn Gly Leu Ser Leu Ile Glu Cys Glu Gly Asn Ile Thr Ile 115 120 125 Pro Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Leu 130 135 140 Leu Gln Tyr Glu Leu Gly Gly Pro Arg Val Cys Val Gln Thr Ala Tyr 145 150 155 160 Gly Ile Glu Val Glu Val Glu Ser Ile Pro Tyr Asp Pro Ser Leu Met 165 170 175 Val Phe Met Asp Tyr Arg Asp Tyr Thr Lys His Lys Ser Gln Ser Leu 180 185 190 Glu Ala Gln Tyr Pro Thr Phe Leu Tyr Val Met Pro Met Ser Pro Thr 195 200 205 Lys Val Phe Phe Glu Glu Thr Cys Leu Ala Ser Lys Glu Ala Met Pro 210 215 220 Phe Glu Leu Leu Lys Thr Lys Leu Met Ser Arg Leu Lys Thr Met Gly 225 230 235 240 Ile Arg Ile Thr Lys Thr Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val 245 250 255 Gly Gly Ser Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala Phe Gly Ala 260 265 270 Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser 275 280 285 Leu Ser Glu Ala Pro Asn Tyr Ala Ala Val Ile Ala Lys Ile Leu Gly 290 295 300 Lys Gly Asn Ser Lys Gln Met Leu Asp His Gly Arg Tyr Thr Thr Asn 305 310 315 320 Ile Ser Lys Gln Ala Trp Glu Thr Leu Trp Pro Leu Glu Arg Lys Arg 325 330 335 Gln Arg Ala Phe Phe Leu Phe Gly Leu Ala Leu Ile Val Gln Met Asp 340 345 350 Ile Glu Gly Thr Arg Thr Phe Phe Arg Thr Phe Phe Arg Leu Pro Thr 355 360 365 Trp Met Trp Trp Gly Phe Leu Gly Ser Ser Leu Ser Ser Thr Asp Leu 370 375 380 Ile Ile Phe Ala Phe Tyr Met Phe Ile Ile Ala Pro His Ser Leu Arg 385 390 395 400 Met Gly Leu Val Arg His Leu Leu Ser Asp Pro Thr Gly Gly Thr Met 405 410 415 Leu Lys Ala Tyr Leu Thr Ile 420 75 422 PRT Lycopersicon esculentum 75 Val Leu Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu Ala Leu 1 5 10 15 Ala Ala Glu Ser Ala Lys Leu Gly Leu Asn Val Gly Leu Val Gly Pro 20 25 30 Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu Asp Glu Phe Lys 35 40 45 Asp Leu Gly Leu Gln Ala Cys Ile Glu His Val Trp Arg Asp Thr Ile 50 55 60 Val Tyr Leu Asp Asp Asp Glu Pro Ile Leu Ile Gly Arg Ala Tyr Gly 65 70 75 80 Arg Val Ser Arg His Phe Leu His Glu Glu Leu Leu Lys Arg Cys Val 85 90 95 Glu Ala Gly Val Leu Tyr Leu Asn Ser Lys Val Asp Arg Ile Val Glu 100 105 110 Ala Thr Asn Gly Gln Ser Leu Val Glu Cys Glu Gly Asp Val Val Ile 115 120 125 Pro Cys Arg Phe Val Thr Val Ala Ser Gly Ala Ala Ser Gly Lys Phe 130 135 140 Leu Gln Tyr Glu Leu Gly Ser Pro Arg Val Ser Val Gln Thr Ala Tyr 145 150 155 160 Gly Val Glu Val Glu Val Asp Asn Asn Pro Phe Asp Pro Ser Leu Met 165 170 175 Val Phe Met Asp Tyr Arg Asp Tyr Leu Arg His Asp Ala Gln Ser Leu 180 185 190 Glu Ala Lys Tyr Pro Thr Phe Leu Tyr Ala Met Pro Met Ser Pro Thr 195 200 205 Arg Val Phe Phe Glu Glu Thr Cys Leu Ala Ser Lys Asp Ala Met Pro 210 215 220 Phe Asp Leu Leu Lys Lys Lys Leu Met Leu Arg Leu Asn Thr Leu Gly 225 230 235 240 Val Arg Ile Lys Glu Ile Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val 245 250 255 Gly Gly Ser Leu Pro Asn Thr Glu Gln Lys Thr Leu Ala Phe Gly Ala 260 265 270 Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser 275 280 285 Leu Ser Glu Ala Pro Lys Cys Ala Ser Val Leu Ala Asn Ile Leu Arg 290 295 300 Gln His Tyr Ser Lys Asn Met Leu Thr Ser Ser Ser Ile Pro Ser Ile 305 310 315 320 Ser Thr Gln Ala Trp Asn Thr Leu Trp Pro Gln Glu Arg Lys Arg Gln 325 330 335 Arg Ser Phe Phe Leu Phe Gly Leu Ala Leu Ile Leu Gln Leu Asp Ile 340 345 350 Glu Gly Ile Arg Ser Phe Phe Arg Ala Phe Phe Arg Val Pro Lys Trp 355 360 365 Met Trp Gln Gly Phe Leu Gly Ser Ser Leu Ser Ser Ala Asp Leu Met 370 375 380 Leu Phe Ala Phe Tyr Met Phe Ile Ile Ala Pro Asn Asp Met Arg Lys 385 390 395 400 Gly Leu Ile Arg His Leu Leu Ser Asp Pro Thr Gly Ala Thr Leu Ile 405 410 415 Arg Thr Tyr Leu Thr Phe 420 76 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 76 Glu Leu Gly Gly 1 77 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 77 Pro Arg Val Ser 1 78 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 78 Tyr Asp Pro Asp 1 79 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 79 Leu Gly Leu Gln 1 80 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 80 Lys Ile Phe Phe 1 81 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 81 Glu Glu Thr Cys 1 82 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 82 Trp Pro Leu Glu 1 83 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 83 Arg Lys Arg Gln 1 84 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 84 Ile Val Leu Met 1 85 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 85 Asp Thr Glu Gly 1 86 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 86 Ile Ile Phe Ala 1 87 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 87 Leu Tyr Met Phe 1 88 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 88 Ala Thr Gly Tyr 1 89 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 89 Ser Val Val Arg 1 90 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 90 Val Ile Ala Glu 1 91 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 91 Ile Leu Arg Gln 1 92 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 92 Gln Ile Asn Ser 1 93 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 93 Asn Ile Ser Lys 1 94 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 94 Arg Gln Arg Ala 1 95 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 95 Phe Phe Leu Phe 1 96 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 96 Phe Asp Thr Glu 1 97 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide 97 Gly Thr Arg Thr 1 

What is claimed:
 1. A chimeric polynucleotide encoding a carotene-synthesizing enzyme, comprising a polynucleotide encoding an N-terminal portion of a first enzyme and a C-terminal portion of a second enzyme.
 2. A chimeric polynucleotide encoding a bicyclic-epsilon-carotenoid synthesizing enzyme, comprising a polynucleotide encoding an N-terminal portion of a first enzyme and a C-terminal portion of a second enzyme.
 3. A chimeric polynucleotide encoding a bicyclic-ε-carotene-synthesizing enzyme, comprising a polynucleotide encoding an N-terminal portion of a lettuce enzyme and a C-terminal portion of an Arabidopsis enzyme.
 4. A chimeric polynucleotide encoding a bicyclic-ε-carotene-synthesizing enzyme, comprising a polynucleotide encoding an N-terminal portion of an Arabidopsis enzyme and a C-terminal portion of lettuce enzyme.
 5. The chimeric polynucleotide as in one of claims 1-4, wherein the enzyme is an ε-cyclase.
 6. The chimeric polynucleotide as in one of claims 1-4, wherein the enzyme has a catalytic domain comprising six amino acids.
 7. The chimeric polynucleotide of claim 6, wherein the first amino acid residue of the six amino acids is alanine (A), serine (S), glutamic acid (E) or asparagine (D); the second amino acid position of the six amino acids is arginine (R), leucine (L), histidine (H) or isoleucine (I); the third amino acid position of the six amino acids is isoleucine (I) or leucine (L); the fourth amino acid position of the six amino acids is valine (V) or leucine (L); the fifth amino acid position of the six amino acids is glutamine (Q), leucine (L) or lysine (K); and the sixth amino acid position of the six amino acids is phenylalanine (F), leucine (L), methionine (M) or leucine (L).
 8. The chimeric polynucleotide of claim 7, wherein the catalytic domain comprises amino acids residues SHIVLM (SEQ ID NO: 41) or SRIVLM (SEQ ID NO: 42).
 9. A ε-cyclase enzyme comprising a catalytic domain of amino acid residues SHIVLM (SEQ ID NO: 41) or SRIVLM (SEQ ID NO: 42).
 10. A method for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprising a) providing a first polynucleotide encoding a full-length enzyme and a second polynucleotide encoding a full-length enzyme, each polynucleotide being subcloned in tandem into a vector; b) providing a first primer for hybridizing to the first polynucleotide and a second primer for hybridizing to the second polynucleotide; c) performing an inverse polymerase chain reaction using the first and the second primer and the vector to obtain a construct containing a chimeric polynucleotide containing a 5′ end of the first polynucleotide and a 3′ end of the second polynucleotide; d) repeating steps b) and c) with a plurality of different first primers and a plurality of different second primers for obtaining a plurality of constructs containing different chimeric polynucleotides for scanning along the encoded amino acid sequence one amino acid at a time; e) transfecting a host cell with one or more of the plurality of constructs and growing the host cell under conditions for expressing chimeric proteins encoded by the chimeric polynucleotides; f) performing enzyme catalysis with the chimeric proteins on an enzyme-specific substrate in the host cell, and g) identifying the enzyme-catalyzing domain encoded by the chimeric proteins by identification of at least one carotenoid compound from the enzyme catalysis of step f).
 11. The method of claim 10, wherein the first polynucleotide encodes an N-terminal portion of a lettuce enzyme and the second polynucleotide encodes a C-terminal portion of an Arabidopsis enzyme.
 12. The method of claim 10, wherein the first polynucleotide encodes an N-terminal portion of an Arabidopsis enzyme and the second polynucleotide encodes a C-terminal portion of a lettuce enzyme.
 13. The method of claim 10, wherein the enzyme is ε-cyclase.
 14. The method of claim 10 or 11, wherein the first primer is a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and the second primer is a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO:
 10. 15. The method of claim 10 or 12, wherein the first primer is a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, and the second primer is a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO:
 20. 16. A method for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprising a) providing a vector containing a polynucleotide encoding the full-length enzyme and a primer for hybridizing to the polynucleotide; b) performing site-directed mutagenesis using the vector and the primer for obtaining a construct containing a site-directed mutant of the polynucleotide encoding the enzyme; c) transfecting a host cell with the construct and growing the host cell under conditions for expressing a site-directed mutant of a protein encoded by the site-directed mutant of the polynucleotide; d) allowing enzyme catalysis with the site-directed mutant of the protein on an enzyme-specific substrate in the host cell; and e) identifying the enzyme-catalyzing domain encoded by the site-directed mutant of the protein by identification of a carotenoid compound from the enzyme catalysis of step d).
 17. A method for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprising a) providing a vector containing a polynucleotide encoding the full-length enzyme and a primer for hybridizing to the polynucleotide; b) performing site-directed mutagenesis using the primer and the vector for obtaining a construct containing a truncated polynucleotide encoding a fragment of the enzyme; c) transfecting a host cell with the construct and growing the host cell under conditions for expressing a truncated protein encoded by the truncated polynucleotide; d) allowing enzyme catalysis with the truncated protein on an enzyme-specific substrate in the host cell; and e) identifying the enzyme-catalyzing domain encoded by the truncated protein by identification of a carotenoid compound from the enzyme catalysis of step d).
 18. A method for producing ε,ε-carotene in an ε,ε-carotene-deficient, lycopene-expressing host, comprising transfecting the host with a chimeric polynucleotide encoding a host-specific ε-cyclase enzyme containing a catalytic domain according to SEQ ID NO: 41 or SEQ ID NO: 42 and expressing the chimeric polynucleotide.
 19. A method for increasing ε,ε-carotene in a lycopene-expressing host, comprising transfecting the host with a chimeric polynucleotide encoding a host-specific ε-cyclase enzyme containing a catalytic domain according to SEQ ID NO: 41 or SEQ ID NO: 42 and expressing the chimeric polynucleotide. 